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 a LANGE medical book

Harper’s
Illustrated
Biochemistry THIRTY-SECOND EDITION

Peter J. Kennelly, PhD Victor W. Rodwell, PhD

Professor Professor (Emeritus) of Biochemistry
Department of Biochemistry Purdue University
Virginia Tech West Lafayette, Indiana
Blacksburg, Virginia
P. Anthony Weil, PhD
Kathleen M. Botham, PhD, DSc Professor Emeritus of Molecular Physiology & Biophysics
Emeritus Professor of Biochemistry Vanderbilt University
Department of Comparative Biomedical Sciences Nashville, Tennessee
Royal Veterinary College
University of London
London, United Kingdom

Owen P. McGuinness, PhD

Professor
Department of Molecular Physiology & Biophysics
Vanderbilt University
School of Medicine
Nashville, Tennessee

New York Chicago San Francisco Athens London Madrid Mexico City
Milan New Delhi Singapore Sydney Toronto
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CoAuthors
David A. Bender, PhD Robert K. Murray, MD, PhD
Proessor (Emeritus) o Nutritional Biochemistry Emeritus Proessor o Biochemistry
University College London University o oronto
London, United Kingdom oronto, Ontario, Canada

Peter L. Gross, MD, MSc, FRCP(C) Margaret L. Rand, PhD

Associate Proessor Senior Associate Scientist
Department o Medicine Division o Hematology/Oncology
McMaster University Hospital or Sick Children, oronto
Hamilton, Ontario, Canada Proessor, Department o Biochemistry
University o oronto, oronto, Canada
January D. Haile, PhD
Associate Proessor o Biochemistry and Molecular Biology Joe Varghese, MD, PhD
Centre College Proessor and Head o Biochemistry
Danville, Kentucky Christian Medical College
Vellore, amil Nadu, India
Molly Jacob, MD, PhD, MNASc, FRCPath
Proessor o Biochemistry
Christian Medical College
Vellore, amil Nadu, India

Peter A. Mayes, PhD, DSc

Emeritus Proessor o Veterinary Biochemistry
Royal Veterinary College
University o London
London, United Kingdom

iii
This page intentionally left blank
Contents
Preace ix

S E C T I O N
10 The Biochemical Roles o Transition
Structures & Functions of Metals 96

I Proteins & Enzymes 1 Peter J. Kennelly, PhD

1 Biochemistry & Medicine 1 S E C T I O N

Victor W. Rodwell, PhD
2 Water & pH 6
Peter J. Kennelly, PhD, & Victor W. Rodwell, PhD
3 Amino Acids & Peptides 15
Peter J. Kennelly, PhD, & Victor W. Rodwell, PhD
4 Proteins: Determination o Primary
Structure 24
Peter J. Kennelly, PhD, & Victor W. Rodwell, PhD
5 Proteins: Higher Orders o Structure 34
Peter J. Kennelly, PhD, & Victor W. Rodwell, PhD
Bioenergetics 109
III
11 Bioenergetics: The Role o ATP 109
Kathleen M. Botham, PhD, DSc, & Peter A. Mayes, PhD, DSc
12 Biologic Oxidation 115
Kathleen M. Botham, PhD, DSc, & Peter A. Mayes, PhD, DSc
13 The Respiratory Chain & Oxidative
Phosphorylation 121
Kathleen M. Botham, PhD, DSc, & Peter A. Mayes, PhD, DSc

S E C T I O N
S E C T I O N Metabolism of
Enzymes: Kinetics,

II Mechanism, Regulation, &

Role of Transition Metals 49
IV Carbohydrates 133

14 Overview o Metabolism & the Provision o

6 Proteins: Myoglobin & Hemoglobin 49 Metabolic Fuels 133
Peter J. Kennelly, PhD, & Victor W. Rodwell, PhD Owen P. McGuinness, PhD

7 Enzymes: Mechanism o Action 59 15 Saccharides (ie, Carbohydrates) o

Peter J. Kennelly, PhD, & Victor W. Rodwell, PhD
8 Enzymes: Kinetics 71
Victor W. Rodwell, PhD
9 Enzymes: Regulation o Activities 85
Peter J. Kennelly, PhD, & Victor W. Rodwell, PhD
Physiological Signicance 147
Owen P. McGuinness, PhD
16 The Citric Acid Cycle: A Pathway Central
to Carbohydrate, Lipid, & Amino Acid
Metabolism 156
Owen P. McGuinness, PhD

v
vi CONTENTS

17 Glycolysis & the Oxidation o Pyruvate 163 28 Catabolism o Proteins & o Amino Acid
Owen P. McGuinness, PhD Nitrogen 279
Victor W. Rodwell, PhD
18 Metabolism o Glycogen 171
Owen P. McGuinness, PhD 29 Catabolism o the Carbon Skeletons o
Amino Acids 290
19 Gluconeogenesis & the Control o Victor W. Rodwell, PhD
Blood Glucose 180
Owen P. McGuinness, PhD 30 Conversion o Amino Acids to Specialized
Products 306
20 The Pentose Phosphate Pathway & Other Victor W. Rodwell, PhD
Pathways o Hexose Metabolism 191
Owen P. McGuinness, PhD 31 Porphyrins & Bile Pigments 315
Victor W. Rodwell, PhD

S E C T I O N

Metabolism of Lipids 205

V S E C T I O N
Structure, Function, &
Replication of Informational
21 Lipids o Physiologic Signicance 205
Kathleen M. Botham, PhD, DSc, & Peter A. Mayes, PhD, DSc
VII Macromolecules 329

32 Nucleotides 329
22 Oxidation o Fatty Acids: Ketogenesis 217 Victor W. Rodwell, PhD
Kathleen M. Botham, PhD, DSc, & Peter A. Mayes, PhD, DSc
33 Metabolism o Purine & Pyrimidine
23 Biosynthesis o Fatty Acids & Nucleotides 337
Eicosanoids 226 Victor W. Rodwell, PhD
Kathleen M. Botham, PhD, DSc, & Peter A. Mayes, PhD, DSc
34 Nucleic Acid Structure & Function 348
24 Metabolism o Acylglycerols & P. Anthony Weil, PhD
Sphingolipids 239
Kathleen M. Botham, PhD, DSc, & Peter A. Mayes, PhD, DSc 35 DNA Organization, Replication, &
Repair 360
25 Lipid Transport & Storage 247 P. Anthony Weil, PhD
Kathleen M. Botham, PhD, DSc, & Peter A. Mayes, PhD, DSc
36 RNA Synthesis, Processing, &
26 Cholesterol Synthesis, Transport, & Modication 384
Excretion 259 P. Anthony Weil, PhD
Kathleen M. Botham, PhD, DSc, & Peter A. Mayes, PhD, DSc
37 Protein Synthesis & the Genetic Code 404
P. Anthony Weil, PhD
S E C T I O N
Metabolism of Proteins &
VI Amino Acids 273 38 Regulation o Gene Expression 420
P. Anthony Weil, PhD

27 Biosynthesis o the Nutritionally 39 Molecular Genetics, Recombinant DNA, &

Nonessential Amino Acids 273 Genomic Technology 444
Victor W. Rodwell, PhD P. Anthony Weil, PhD
 CONTENTS vii

S E C T I O N S E C T I O N
Biochemistry of
Extracellular & Intracellular Special Topics (B) 581
VIII Communication 467 X
49 Intracellular Trafc & Sorting o Proteins 581
40 Membranes: Structure &
Kathleen M. Botham, PhD, DSc, & Robert K. Murray, MD, PhD
Function 467
P. Anthony Weil, PhD
50 The Extracellular Matrix 599
Kathleen M. Botham, PhD, DSc, & Robert K. Murray, MD, PhD
41 The Diversity o the Endocrine
System 488
P. Anthony Weil, PhD
51 Muscle & the Cytoskeleton 618
January D. Haile, PhD, & Peter J. Kennelly, PhD

42 Hormone Action & Signal

52 Plasma Proteins & Immunoglobulins 634
Transduction 508
Peter J. Kennelly, PhD
P. Anthony Weil, PhD

53 Red Blood Cells 653

Peter J. Kennelly, PhD

S E C T I O N
54 White Blood Cells 664
Special Topics (A) 527
IX Peter J. Kennelly, PhD

43 Nutrition, Digestion, & S E C T I O N

Absorption 527 Special Topics (C) 677
David A. Bender, PhD
XI
44 Micronutrients: Vitamins &
Minerals 535 55 Hemostasis & Thrombosis 677
Peter L. Gross, MD, MSc, FRCP(C),
David A. Bender, PhD
P. Anthony Weil, PhD, & Margaret L. Rand, PhD

45 Free Radicals & Antioxidant 56 Cancer: An Overview 689

Nutrients 549 Molly Jacob, MD, PhD, MNASc, FRCPath,
David A. Bender, PhD Joe Varghese, MD, PhD, & P. Anthony Weil, PhD

46 Glycoproteins 554 57 The Biochemistry o Aging 717

David A. Bender, PhD Peter J. Kennelly, PhD

47 Metabolism o Xenobiotics 564 58 Biochemical Case Histories 729

David A. Bender, PhD & Robert K. Murray, MD, PhD David A. Bender, PhD

48 Clinical Biochemistry 568 The Answer Bank 741

David A. Bender, PhD Index 745
This page intentionally left blank
Preace
Te authors and publishers are pleased to present the thirty-
second edition o Harper’s Illustrated Biochemistry. Te rst
edition, entitled Harper’s Biochemistry, was published in
1939 under the sole authorship o Dr Harold Harper at the
University o Caliornia School o Medicine, San Francisco,
Caliornia. Presently entitled Harper’s Illustrated Biochemistry,
the book continues, as originally intended, to provide a con-
cise survey o aspects o biochemistry most relevant to the
study o medicine. Various authors have contributed to sub-
sequent editions o this medically oriented biochemistry text,
which is now observing its 83rd year.
Cover Illustration for
the Thirty-Second Edition
Te global COVID-19 pandemic has provided a dramatic,
ace-to-ace demonstration o both the power and limita-
tions o molecular medicine and epidemiology. Te rapid
development o highly eective vaccines was made possible
by the adaptation o novel RNA-based approaches in which
the patient’s immune response is activated via the endogenous
expression o genetically-encoded antigens, rather than the
physical injection o a non-inectious antigen. Utilizing the
patient’s own cells as the bioreactor or generating antigens,
rather than some animal or culture, enabled scientists to use
the sel-ampliying capacity o polynucleotides to accelerate
both the speed o vaccine development and subsequent large-
scale manuacture. Te illustration on the cover o the thirty-
second edition depicts a neutralizing antibody, in blue, bound
to the spike protein on the surace o the SARS-CoV-2 coro-
navirus, better known as COVID-19, which is shown in red.
Te epitope to which the antibody binds overlaps that at which
the virus binds to the ACE-2 receptor, the membrane protein
by which the pathogen recognizes, binds to, and subsequently
invades human cells. Terapeutic antibodies thus protect by
physically blocking association o the Spike protein with the
ACE-2 receptor.
Changes in the Thirty-Second Edition
As always, Harper’s Illustrated Biochemistry continues to
emphasize the close relationship o biochemistry to the under-
standing o diseases, their pathology, and the practice o medi-
cine. With the retirement o long-time contributor David A.
Bender, Pro. Owen P. McGuinness o Vanderbilt University
has joined as a new coauthor. In addition to the resh per-
spectives and novel insights provided by Pro. McGuinness,
the contents o most chapters have been updated and provide
the reader with the most current and pertinent inormation.
For example, in Chapter 6 the description o the Bohr eect’s
contributions to CO2 transport and release rom the lungs has
been reorganized and expanded, while Chapter 9 has been
updated and reorganized to include expanded coverage o
zymogen activation in enzyme regulation.
Organization of the Book
All 58 chapters o the thirty-second edition place major emphasis
on the medical relevance o biochemistry. opics are organized
under 11 major headings. In order to assist study and to acilitate
retention o the contained inormation, Questions ollow each
Section. An Answer Bank ollows Chapter 58.
Section I includes a brie history o biochemistry and
emphasizes the interrelationships between biochemistry
and medicine. Water and the importance o homeostasis
o intracellular pH are reviewed, and the various orders
o proteins structure are addressed.
Section II begins with a chapter on hemoglobin. Te
next our chapters address the mechanism o action,
kinetics, metabolic regulation o enzymes, and the
role o metal ions in multiple aspects o intermediary
metabolism.
Section III addresses bioenergetics and the role o
high-energy phosphates in energy capture and transer,
the oxidation–reduction reactions involved in biologic
oxidation, and metabolic details o energy capture via
the respiratory chain and oxidative phosphorylation.
Section IV considers the metabolism o carbohydrates
via glycolysis, the citric acid cycle, the pentose phosphate
pathway, glycogen metabolism, gluconeogenesis, and the
control o blood glucose.
Section V outlines the nature o simple and complex
lipids, lipid transport and storage, the biosynthesis and
degradation o atty acids and more complex lipids, and
the reactions and metabolic regulation o cholesterol
biosynthesis and transport in human subjects.
Section VI discusses protein catabolism, urea
biosynthesis, and the catabolism o amino acids, and
stresses the medically signicant metabolic disorders
associated with their incomplete catabolism. Te nal
chapter in this section considers the biochemistry o the
porphyrins and bile pigments.
Section VII rst outlines the structure and unction o
nucleotides and nucleic acids, and then details DNA
replication and repair, RNA synthesis and modication,
protein synthesis, the principles o recombinant DNA
technology, and the regulation o gene expression.
ix
x PREFACE
Section VIII considers aspects o extracellular and
intracellular communication. Specic topics include
membrane structure and unction, the molecular bases
o the actions o hormones, and signal transduction.
Sections IX, X, and XI address many topics o
signicant medical importance.
Section IX discusses nutrition, digestion, and absorption,
micronutrients including, vitamins, ree radicals
and antioxidants, glycoproteins, the metabolism o
xenobiotics, and clinical biochemistry.
Section X addresses intracellular trafc and the sorting
o proteins, the extracellular matrix, muscle and the
cytoskeleton, plasma proteins and immunoglobulins,
and the biochemistry o red cells and o white cells.
Section XI includes hemostasis and thrombosis, an
overview o cancer, the biochemistry o aging, and a
selection o case histories.
Acknowledgments
Te authors thank Michael Weitz or his role in the planning
o this edition and Peter Boyle or overseeing its preparation
or publication. We also thank asneem Kauser and her col-
leagues at KnowledgeWorks Global Ltd. or their eorts in
managing editing, typesetting, and artwork. We grateully
acknowledge numerous suggestions and corrections received
rom students and colleagues rom around the world.
Peter J. Kennelly
Kathleen M. Botham
Owen P. McGuinness
Victor W. Rodwell
P. Anthony Weil
 S E C T I O N
Structures & Functions
I of Proteins & Enzymes

C H A P T E R

Biochemistry & Medicine

Victor W. Rodwell, PhD
OBJ E C TI VE S
After studying this chapter,
you should be able to:
1
■ Understand the importance of the ability of cell-free extracts of yeast to
ferment sugars, an observation that enabled discovery of the intermediates of
fermentation, glycolysis, and other metabolic pathways.
■ Appreciate the scope of biochemistry and its central role in the life sciences,
and that biochemistry and medicine are intimately related disciplines.
■ Appreciate that biochemistry integrates knowledge of the chemical processes
in living cells with strategies to maintain health, understand disease, identify
potential therapies, and enhance our understanding of the origins of life on
earth.
■ Describe how genetic approaches have been critical for elucidating many areas
of biochemistry, and how the Human Genome Project has furthered advances
in numerous aspects of biology and medicine.

BIOMEDICAL IMPORTANCE DISCOVERY THAT A CELL-FREE

Biochemistry and medicine enjoy a mutually cooperative
relationship. Biochemical studies have illuminated many
aspects o health and disease, and the study o various aspects
o health and disease has opened up new areas o biochem-
istry. he medical relevance o biochemistry both in normal
and abnormal situations is emphasized throughout this book.
Biochemistry makes signiicant contributions to the ields o
cell biology, physiology, immunology, microbiology, pharma-
cology, toxicology, and epidemiology, as well as the ields o
inlammation, cell injury, and cancer. hese close relationships
emphasize that lie, as we know it, depends on biochemical
reactions and processes.
EXTRACT OF YEAST CAN
FERMENT SUGAR
Although the ability o yeast to “erment” various sugars
to ethyl alcohol has been known or millennia, only com-
paratively recently did this process initiate the science o
biochemistry. he great French microbiologist Louis Pasteur
maintained that ermentation could only occur in intact cells.
However, in 1899, the brothers Büchner discovered that er-
mentation could occur in the absence o intact cells when they
stored a yeast extract in a crock o concentrated sugar solu-
tion, added as a preservative. Overnight, the contents o the
crock ermented, spilled over the laboratory bench and loor,

1
2 SECTION I Structures & Functions of Proteins & Enzymes
and dramatically demonstrated that ermentation can proceed
in the absence o an intact cell. his discovery unleashed an
avalanche o research that initiated the science o biochemis-
try. Investigations revealed the vital roles o inorganic phos-
phate, ADP, AP, and NAD(H), and ultimately identiied
the phosphorylated sugars and the chemical reactions and
enzymes that convert glucose to pyruvate (glycolysis) or to
ethanol and CO2 (ermentation). Research beginning in the
1930s identiied the intermediates o the citric acid cycle and
o urea biosynthesis, and revealed the essential roles o certain
vitamin-derived coactors or “coenzymes” such as thiamin
pyrophosphate, ribolavin, and ultimately coenzyme A, coen-
zyme Q, and cobamide coenzyme. he 1950s revealed how
complex carbohydrates are synthesized rom, and broken
down into simple sugars, and the pathways or biosynthesis
o pentoses, and the catabolism o amino acids and atty acids.
Investigators employed animal models, perused intact
organs, tissue slices, cell homogenates and their subractions,
and subsequently puriied enzymes. Advances were enhanced
by the development o analytical ultracentriugation, paper
and other orms o chromatography, and the post-World
War II availability o radioisotopes, principally 14C, 3H, and 32P,
as “tracers” to identiy the intermediates in complex pathways
such as that o cholesterol biosynthesis. X-ray crystallogra-
phy was then used to solve the three-dimensional structures
o numerous proteins, polynucleotides, enzymes, and viruses.
Genetic advances that ollowed the realization that DNA was a
double helix include the polymerase chain reaction, and trans-
genic animals or those with gene knockouts. he methods used
to prepare, analyze, puriy, and identiy metabolites and the
activities o natural and recombinant enzymes and their three-
dimensional structures are discussed in the ollowing chapters.
BIOCHEMISTRY & MEDICINE
HAVE PROVIDED MUTUAL
ADVANCES
he two major concerns or workers in the health sciences—
and particularly physicians—are the understanding and
maintenance o health and eective treatment o disease. Bio-
chemistry impacts both o these undamental concerns, and
Biochemistry
Nucleic
acids Proteins
Genetic Sickle cell
diseases anemia
Medicine
FIGURE 1–1 A two-way street connects biochemistry and medicine. Knowledge of the biochemical topics listed above the green
line of the diagram has clarified our understanding of the diseases shown below the green line. Conversely, analyses of the diseases have cast
light on many areas of biochemistry. Note that sickle cell anemia is a genetic disease, and that both atherosclerosis and diabetes mellitus have
genetic components.
the interrelationship o biochemistry and medicine is a wide,
two-way street. Biochemical studies have illuminated many
aspects o health and disease, and conversely, the study o vari-
ous aspects o health and disease has opened up new areas o
biochemistry (Figure 1–1). An early example o how investiga-
tion o protein structure and unction revealed the single di-
erence in amino acid sequence between normal hemoglobin
and sickle cell hemoglobin. Subsequent analysis o numerous
variant sickle cell and other hemoglobins has contributed sig-
niicantly to our understanding o the structure and unction
both o hemoglobin and o other proteins. During the early
1900s, the English physician Archibald Garrod studied patients
with the relatively rare disorders o alkaptonuria, albinism, cys-
tinuria, and pentosuria, and established that these conditions
were genetically determined. Garrod designated these condi-
tions as inborn errors of metabolism. His insights provided
a oundation or the development o the ield o human bio-
chemical genetics. A more recent example was investigation
o the genetic and molecular basis o amilial hypercholester-
olemia, a disease that results in early-onset atherosclerosis. In
addition to clariying dierent genetic mutations responsible
or this disease, this provided a deeper understanding o cell
receptors and mechanisms o uptake, not only o cholesterol
but also o how other molecules cross cell membranes. Stud-
ies o oncogenes and tumor suppressor genes in cancer cells
have directed attention to the molecular mechanisms involved
in the control o normal cell growth. hese examples illustrate
how the study o disease can open up areas o basic biochemi-
cal research. Science provides physicians and other workers
in health care and biology with a oundation that impacts
practice, stimulates curiosity, and promotes the adoption o
scientiic approaches or continued learning.
BIOCHEMICAL PROCESSES
UNDERLIE HUMAN HEALTH
Biochemical Research Impacts
Nutrition & Preventive Medicine
he World Health Organization (WHO) deines health as a state
o “complete physical, mental, and social well-being and not
merely the absence o disease and inirmity.” From a biochemical
Lipids Carbohydrates
Athero- Diabetes
sclerosis mellitus

viewpoint, health may be considered that situation in which all
o the many thousands o intra- and extracellular reactions that
occur in the body are proceeding at rates commensurate with
the organism’s survival under pressure rom both internal and
external challenges. he maintenance o health requires optimal
dietary intake o vitamins, certain amino acids and fatty acids,
various minerals, and water. Understanding nutrition depends
to a great extent on knowledge o biochemistry, and the sciences
o biochemistry and nutrition share a ocus on these chemicals.
Recent increasing emphasis on systematic attempts to maintain
health and orestall disease, or preventive medicine, includes
nutritional approaches to the prevention o diseases such as
atherosclerosis and cancer.
Most Diseases Have a Biochemical Basis
Apart rom inectious organisms and environmental pollut-
ants, many diseases are maniestations o abnormalities in genes,
proteins, chemical reactions, or biochemical processes, each
o which can adversely aect one or more critical biochemical
unctions. Examples o disturbances in human biochemistry
responsible or diseases or other debilitating conditions include
electrolyte imbalance, deective nutrient ingestion or absorp-
tion, hormonal imbalances, toxic chemicals or biologic agents,
and DNA-based genetic disorders. o address these challenges,
biochemical research continues to be interwoven with studies in
disciplines such as genetics, cell biology, immunology, nutrition,
pathology, and pharmacology. In addition, many biochemists are
vitally interested in contributing to solutions to key issues such
as the ultimate survival o mankind, and educating the public to
support use o the scientiic method in solving environmental
and other major problems that conront our civilization.
Impact of the Human Genome Project
on Biochemistry, Biology, & Medicine
Initially unanticipated rapid progress in the late 1990s in
sequencing the human genome led in the mid-2000s to the
Transcriptomics Proteomics
Metabolomics
Pharmacogenomics
HGP
(Genomics)
Bioengineering
Biophysics
Stem cell biology
Nanotechnology
Molecular diagnostics
FIGURE 1–2 The Human Genome Project (HGP) has influenced many disciplines and areas of research. Biochemistry is not listed
since it predates commencement of the HGP, but disciplines such as bioinformatics, genomics, glycomics, lipidomics, metabolomics, molecular
diagnostics, proteomics, and transcriptomics are nevertheless active areas of biochemical research.
CHAPTER 1 Biochemistry & Medicine 3
announcement that over 90% o the genome had been sequenced.
his eort was headed by the International Human Genome
Sequencing Consortium and by Celera Genomics. Except or
a ew gaps, the sequence o the entire human genome was
completed in 2003, just 50 years ater the description o the
double-helical nature o DNA by Watson and Crick. he
implications or biochemistry, medicine, and indeed or all
o biology, are virtually unlimited. For example, the ability
to isolate and sequence a gene and to investigate its structure
and unction by sequencing and “gene knockout” experi-
ments have revealed previously unknown genes and their
products, and new insights have been gained concerning
human evolution and procedures or identiying disease-
related genes.
Major advances in biochemistry and understanding
human health and disease continue to be made by mutation
o the genomes o model organisms such as yeast, the ruit
ly Drosophila melanogaster, the roundworm Caenorhabditis
elegans, and the zebra ish; all organisms that can be geneti-
cally manipulated to provide insight into the unctions o
individual genes. hese advances can potentially provide
clues to curing human diseases such as cancer and Alzheimer
disease. Figure 1–2 highlights areas that have developed or
accelerated as a direct result o progress made in the Human
Genome Project (HGP). New “-omics” ields ocus on com-
prehensive study o the structures and unctions o the mol-
ecules with which each is concerned. he products o genes
(RNA molecules and proteins) are being studied using the
techniques o transcriptomics and proteomics. A spectacu-
lar example o the speed o progress in transcriptomics is the
explosion o knowledge about small RNA molecules as regu-
lators o gene activity. Other -omics ields include glycomics,
lipidomics, metabolomics, nutrigenomics, and pharma-
cogenomics. o keep pace with the inormation generated,
bioinformatics has received much attention. Other related
ields to which the impetus rom the HGP has carried over are
biotechnology, bioengineering, biophysics, and bioethics.
Glycomics Lipidomics
Nutrigenomics
Bioinformatics
Biotechnology
Bioethics
Gene therapy
Systems biology Synthetic biology
4 SECTION I Structures & Functions of Proteins & Enzymes
Deinitions o these -omics ields and other terms appear in
the Glossary o this chapter. Nanotechnology is an active area,
which, or example, may provide novel methods o diagnosis
and treatment or cancer and other disorders. Stem cell biol-
ogy is at the center o much current research. Gene therapy
has yet to deliver the promise that it appears to oer, but it
seems probable that ultimately will occur. Many new molecu-
lar diagnostic tests have developed in areas such as genetic,
microbiologic, and immunologic testing and diagnosis.
Systems biology is also burgeoning. he outcomes o research
in the various areas mentioned above will impact tremen-
dously the uture o biology, medicine, and the health sciences.
Synthetic biology oers the potential or creating living organ-
isms, initially small bacteria, rom genetic material in vitro
that might carry out speciic tasks such as cleansing petroleum
spills. All o the above make the 21st century an exhilarating
time to be directly involved in biology and medicine.
SUMMARY
■ Biochemistry is the science concerned with the molecules
present in living organisms, individual chemical reactions and
their enzyme catalysts, and the expression and regulation o
each metabolic process. Biochemistry has become the basic
language o all biologic sciences.
■ Despite the ocus on human biochemistry in this text,
biochemistry concerns the entire spectrum o lie orms, rom
viruses, bacteria, and plants to complex eukaryotes such as
human beings.
■ Biochemistry, medicine, and other health care disciplines
are intimately related. Health in all species depends on a
harmonious balance o the biochemical reactions occurring in
the body, while disease reects abnormalities in biomolecules,
biochemical reactions, or biochemical processes.
■ Advances in biochemical knowledge have illuminated many
areas o medicine, and the study o diseases has ofen revealed
previously unsuspected aspects o biochemistry.
■ Biochemical approaches are ofen undamental in illuminating
the causes o diseases and in designing appropriate therapy.
Biochemical laboratory tests also represent an integral
component o diagnosis and monitoring o treatment.
■ A sound knowledge o biochemistry and o other related basic
disciplines is essential or the rational practice o medicine and
related health sciences.
■ Results o the HGP and o research in related areas will have
a proound inuence on the uture o biology, medicine, and
other health sciences.
■ Genomic research on model organisms such as yeast, the ruit
y D. melanogaster, the roundworm C. elegans, and the zebra
sh provides insight into understanding human diseases.
GLOSSARY
Bioengineering: Te application o engineering to biology and
medicine.
Bioethics: Te area o ethics that is concerned with the application
o moral and ethical principles to biology and medicine.
Bioinformatics: Te discipline concerned with the collection,
storage, and analysis o biologic data, or example, DNA, RNA,
and protein sequences.
Biophysics: Te application o physics and its techniques to biology
and medicine.
Biotechnology: Te eld in which biochemical, engineering, and
other approaches are combined to develop biologic products o
use in medicine and industry.
Gene Terapy: Applies to the use o genetically engineered genes to
treat various diseases.
Genomics: Te genome is the complete set o genes o an organism,
and genomics is the in-depth study o the structures and
unctions o genomes.
Glycomics: Te glycome is the total complement o simple and
complex carbohydrates in an organism. Glycomics is the
systematic study o the structures and unctions o glycomes
such as the human glycome.
Lipidomics: Te lipidome is the complete complement o lipids
ound in an organism. Lipidomics is the in-depth study o the
structures and unctions o all members o the lipidome and
their interactions, in both health and disease.
Metabolomics: Te metabolome is the complete complement o
metabolites (small molecules involved in metabolism) present
in an organism. Metabolomics is the in-depth study o their
structures, unctions, and changes in various metabolic states.
Molecular Diagnostics: Reers to the use o molecular approaches such
as DNA probes to assist in the diagnosis o various biochemical,
genetic, immunologic, microbiologic, and other medical conditions.
Nanotechnology: Te development and application to medicine
and to other areas o devices such as nanoshells, which are only a
ew nanometers in size (10–9 m = 1 nm).
Nutrigenomics: Te systematic study o the eects o nutrients on
genetic expression and o the eects o genetic variations on the
metabolism o nutrients.
Pharmacogenomics: Te use o genomic inormation and
technologies to optimize the discovery and development o new
drugs and drug targets.
Proteomics: Te proteome is the complete complement o proteins
o an organism. Proteomics is the systematic study o the
structures and unctions o proteomes and their variations in
health and disease.
Stem Cell Biology: Stem cells are undierentiated cells that have the
potential to sel-renew and to dierentiate into any o the adult
cells o an organism. Stem cell biology concerns the biology o
stem cells and their potential or treating various diseases.
Synthetic Biology: Te eld that combines biomolecular techniques
with engineering approaches to build new biologic unctions and
systems.
Systems Biology: Te eld concerns complex biologic systems
studied as integrated entities.
ranscriptomics: Te comprehensive study o the transcriptome,
the complete set o RNA transcripts produced by the genome
during a xed period o time.
APPENDIX
Shown are selected examples o databases that assemble, annotate,
and analyze data o biomedical importance.
ENCODE: ENCyclopedia Of DNA Elements. A collaborative eort
that combines laboratory and computational approaches to
identiy every unctional element in the human genome.

GenBank: Protein sequence database o the National Institutes o
Health (NIH) stores all known biologic nucleotide sequences and
their translations in a searchable orm.
HapMap: Haplotype Map, an international eort to identiy single
nucleotide polymorphisms (SNPs) associated with common
human diseases and dierential responses to pharmaceuticals.
CHAPTER 1 Biochemistry & Medicine 5
ISDB: International Sequence DataBase that incorporates DNA
databases o Japan and o the European Molecular Biology
Laboratory (EMBL).
PDB: Protein DataBase. Tree-dimensional structures o proteins,
polynucleotides, and other macromolecules, including proteins
bound to substrates, inhibitors, or other proteins.
 C H A P T E R

Water & pH
Peter J. Kennelly, PhD, & Victor W. Rodwell, PhD
OBJ E C TI VE S
After studying this chapter,
you should be able to:
2
■ Describe the properties of water that account for its surface tension, viscosity,
liquid state at ambient temperature, and solvent power.
■ Represent the structures of organic compounds that can serve as hydrogen
bond donors or acceptors.
■ Explain the role played by entropy in the association and orientation, in an
aqueous environment, of hydrophobic and amphipathic molecules.
■ Indicate the quantitative contributions of salt bridges, hydrophobic
interactions, and van der Waals forces to stabilizing the 3-D conformation of
macromolecules.
■ Explain the relationship of pH to acidity, alkalinity, and the quantitative
determinants that characterize weak and strong acids.
■ Calculate the shift in pH that accompanies the addition of a given quantity of
acid or base to a buffered solution.
■ Describe what buffers do, how they do it, and the conditions under which
a buffer is most effective under physiologic or other conditions.
■ Use the Henderson-Hasselbalch equation to calculate the net charge on
a polyelectrolyte at a given pH.

BIOMEDICAL IMPORTANCE
Water is the predominant chemical component of living
organisms. Its unique physical properties, which include
the ability to solvate a wide range of organic and inorganic
molecules, derive from water’s dipolar structure and excep-
tional capacity for forming hydrogen bonds. The manner in
which water interacts with a solvated biomolecule influences
the structure both of the biomolecule and of water itself. An
excellent nucleophile, water is a reactant or product in many
metabolic reactions. Regulation of water balance depends on
hypothalamic mechanisms that control thirst, on antidiuretic
hormone (ADH), on retention or excretion of water by the
kidneys, and on evaporative loss. Nephrogenic diabetes insipi-
dus, which involves the inability to concentrate urine or adjust
to subtle changes in extracellular fluid osmolarity, results from
the unresponsiveness of renal tubular osmoreceptors to ADH.
Water has a slight propensity to dissociate into hydroxide
ions and protons. The concentration of protons, or acidity, of
aqueous solutions is generally reported using the logarithmic
pH scale. Bicarbonate and other buffers normally maintain
the pH of extracellular fluid between 7.35 and 7.45. Suspected
disturbances of acid-base balance are verified by measuring
the pH of arterial blood and the CO2 content of venous blood.
Causes of acidosis (blood pH <7.35) include diabetic ketosis
and lactic acidosis. Alkalosis (pH >7.45) may follow vomiting
of acidic gastric contents.
WATER IS AN IDEAL BIOLOGIC
SOLVENT
Water Molecules Form Dipoles
A water molecule is an irregular, slightly skewed tetrahe-
dron with oxygen at its center (Figure 2–1). The corners are
occupied by the two hydrogens and the unshared electrons
of the remaining two sp3-hybridized orbitals of oxygen. The
105° angle between the two hydrogen atoms differs slightly
from the ideal tetrahedral angle, 109.5°. The strongly electro-
negative oxygen atom in a water molecule attracts electrons
away from the hydrogen nuclei, leaving them with a partial

6
 CHAPTER 2 Water & pH 7

H
CH3 CH2 O H O
2e H

2e
H
H
CH3 CH2 O H O
105°
CH2 CH3
H

FIGURE 2–1 The water molecule has tetrahedral geometry. R R II

C O H N

positive charge, while its two unshared electron pairs consti-
tute a region of local negative charge. This asymmetric charge
distribution is referred to as a dipole.
Water’s strong dipole is responsible for its high dielectric
constant. As described quantitatively by Coulomb’s law, the
strength of interaction F between oppositely charged particles
is inversely proportionate to the dielectric constant ε of the
surrounding medium. The dielectric constant for a vacuum
is essentially unity; for hexane it is 1.9; for ethanol, 24.3; and
for water at 25°C, 78.5. When dissolved in water, the force
of attraction between charged and polar species is greatly
decreased relative to solvents with lower dielectric constants.
Its strong dipole and high dielectric constant enable water to
dissolve large quantities of charged compounds such as salts.
RI R III
FIGURE 2–3 Additional polar groups participate in hydrogen
bonding. Shown are hydrogen bonds formed between alcohol and
water, between two molecules of ethanol, and between the peptide
carbonyl oxygen and the peptide nitrogen hydrogen of an adjacent
amino acid.
can participate in hydrogen bonding. The oxygen atoms of
aldehydes, ketones, and amides, for example, provide lone
pairs of electrons that can serve as hydrogen acceptors. Alco-
hols, carboxylic acids, and amines can serve both as hydrogen
acceptors and as donors of unshielded hydrogen atoms for
formation of hydrogen bonds (Figure 2–3).

Water Molecules Form Hydrogen Bonds INTERACTION WITH WATER

A partially unshielded hydrogen nucleus covalently bound to INFLUENCES THE STRUCTURE OF
an electron-withdrawing oxygen or nitrogen atom can interact BIOMOLECULES
with an unshared electron pair on another oxygen or nitrogen
atom to form a hydrogen bond. Since water molecules con- Covalent & Noncovalent Bonds
tain both of these features, hydrogen bonding favors the self- Stabilize Biologic Molecules
association of water molecules into ordered arrays (Figure 2–2).
The covalent bond is the strongest force that holds mol-
On average, each molecule in liquid water associates through
ecules together (Table 2–1). Noncovalent forces, while of
hydrogen bonds with 3.5 others. These bonds are both rela-
lesser magnitude, predominate in stabilizing the folding of
tively weak and transient, with a half-life of a few picoseconds.
the polypeptides and other macromolecules into the complex
Rupture of a hydrogen bond in liquid water requires only about
three-dimensional conformations essential to their functional
4.5 kcal/mol, less than 5% of the energy required to rupture a
competence (see Chapter 5) as well as the association of bio-
covalent O—H bond. The exceptional capacity of this relatively
molecules into multicomponent complexes. Examples of the
small, 18 g/mol, molecule to form hydrogen bonds profoundly
latter include the coalescence of the polypeptide subunits that
influences the physical properties of water and accounts for its
form the hemoglobin tetramer (see Chapter 6); the association
high viscosity, surface tension, and boiling point.
Hydrogen bonding enables water to dissolve many
TABLE 2−1 Bond Energies for Atoms of Biologic
organic biomolecules that contain functional groups which
Significance

H H H H Energy Energy
O O Bond Type (kcal/mol) Bond Type (kcal/mol)
H
H H H O O—O 34 —O
O— 96
O H O
H O H H S—S 51 C—H 99
H O C—N 70 —S
C— 108
H
S—H 81 O—H 110
FIGURE 2–2 Water molecules self-associate via hydrogen
C—C 82 —C
C— 147
bonds. Shown are the association of two water molecules (left) and a
hydrogen-bonded cluster of four water molecules (right). Notice that C—O 84 —N
C— 147
water can serve simultaneously both as a hydrogen donor and as a
N—H 94 —O
C— 164
hydrogen acceptor.
8 SECTION I Structures & Functions of Proteins & Enzymes
of the two polynucleotide strands that comprise a DNA double
helix (see Chapter 34); and the coalescence of billions of phos-
pholipid, glycosphingolipid, cholesterol, and other molecules
into the bilayer that constitutes the foundation of the plasma
membrane of an animal cell (see Chapter 40). These forces,
which can be either attractive or repulsive, involve interactions
both within the biomolecule and, most importantly, between
it and the water that forms the principal component of the sur-
rounding environment.
In Water, Biomolecules Fold to Position
Hydrophobic Groups Within Their
Interior
Most biomolecules are amphipathic; that is, they possess
regions rich in charged or polar functional groups as well as
regions with hydrophobic character. Proteins tend to fold with
the R-groups of amino acids with hydrophobic side chains in
the interior. Amino acids with charged or polar amino acid
side chains (eg, arginine, glutamate, serine; see Table 3–1)
generally are present on the surface in contact with water. A
similar pattern prevails in a phospholipid bilayer where the
charged “head groups” of phosphatidylserine or phosphatidyl-
ethanolamine contact water while their hydrophobic fatty acyl
side chains cluster together, excluding water (see Figure 40–5).
This pattern minimizes energetically unfavorable contacts
between water and hydrophobic groups. It also maximizes
the opportunities for the formation of energetically favorable
charge-dipole, dipole-dipole, and hydrogen bonding interac-
tions between polar groups on the biomolecule and water.
Hydrophobic Interactions
Hydrophobic interaction refers to the tendency of nonpolar
compounds to self-associate in an aqueous environment. This
self-association is driven neither by mutual attraction nor by
what are sometimes incorrectly referred to as “hydrophobic
bonds.” Self-association minimizes the disruption of energeti-
cally favorable interactions between and is therefore driven by
the surrounding water molecules.
While the hydrogen atoms of nonpolar groups such as
the methylene groups of hydrocarbons do not form hydrogen
bonds, they do affect the structure of the water with which
they are in contact. Water molecules adjacent to a hydropho-
bic group are restricted in the number of orientations (degrees
of freedom) that permit them to participate in the maximum
number of energetically favorable hydrogen bonds. Maximal
formation of multiple hydrogen bonds, which maximizes
enthalpy, can be maintained only by increasing the order of
the adjacent water molecules, with an accompanying decrease
in entropy.
It follows from the second law of thermodynamics that the
optimal free energy of a hydrocarbon-water mixture is a func-
tion of both maximal enthalpy (from hydrogen bonding) and
highest entropy (maximum degrees of freedom). Thus, non-
polar molecules tend to form droplets that minimize exposed
FIGURE 2–4 Hydrophobic interactions are driven by the
surrounding water molecules. Water molecules are represented by
one red (oxygen) and two blue (hydrogen) circles. The hydrophobic
surfaces of solute molecules are colored gray and, where present,
hydrophilic ones are colored green. A. When the six hydrophobic
cubes shown are dispersed in water (left), the surrounding water
molecules (red oxygens and blue hydrogens) are forced to engage in
entropically unfavorable interactions with all 36 faces of the cubes.
However, when the six hydrophobic cubes aggregate together
(right), the number of exposed faces is reduced to 22. The aggregate
forms and its stability is maintained, not by some attractive force, but
because aggregation reduces the number of water molecules that
are unfavorably affected by nearly 40%. B. Amphipathic molecules
associate together for the same reason. However, the structure of the
resulting complex (eg, micelle or bilayer) is determined by the geom-
etries of the hydrophobic (gray) and hydrophilic (green) regions.
surface area and reduce the number of water molecules whose
motional freedom becomes restricted (Figure 2–4). Similarly,
in the aqueous environment of the living cell the hydrophobic
portions of amphipathic biopolymers tend to be buried inside
the structure of the molecule, or within a lipid bilayer, mini-
mizing contact with water.
Electrostatic Interactions
Electrostatic interactions between oppositely charged groups
within or between biomolecules are termed salt bridges. Salt
bridges are comparable in strength to hydrogen bonds but act
over larger distances. They therefore often facilitate the binding
of charged molecules and ions to proteins and nucleic acids.
van der Waals Forces
van der Waals forces arise from attractions between transient
dipoles generated by the rapid movement of electrons in all
neutral atoms. Significantly weaker than hydrogen bonds
but potentially extremely numerous, van der Waals forces
decrease as the sixth power of the distance separating atoms
(Figure 2–5). Thus, they act over very short distances, typi-
cally 2 to 4 Å.
Multiple Forces Stabilize Biomolecules
The DNA double helix illustrates the contribution of multiple
forces to the structure of biomolecules. While each individual
DNA strand is held together by covalent bonds, the two strands
of the helix are held together exclusively by noncovalent

.50
Interaction energy (kcaI mol–1)
.25
0 ceed toward its equilibrium point. In the cell, macromolecular
A
–0.25
–0.50
3.0 4.0 5.0
R (Å)
FIGURE 2–5 The strength of van der Waals interactions
varies with the distance, R, between interacting species. The force
of interaction between interacting species increases with decreasing
distance between them until they are separated by the van der Waals
contact distance (see arrow marked A). Repulsion due to interaction
between the electron clouds of each atom or molecule then super-
venes. While individual van der Waals interactions are extremely
weak, their cumulative effect is nevertheless substantial for macro-
molecules such as DNA and proteins which have many atoms in close
contact.
interactions such as hydrogen bonds between nucleotide
bases (Watson-Crick base pairing) and van der Waals interac-
tions between the stacked purine and pyrimidine bases. The
double helix presents the charged phosphate groups and polar
hydroxyl groups from the ribose sugars of the DNA backbone
to water while burying the relatively hydrophobic nucleotide
bases inside. The extended backbone maximizes the distance
between negatively charged phosphates, minimizing unfavor-
able electrostatic interactions (see Figure 34–2).
WATER IS AN EXCELLENT
NUCLEOPHILE
Metabolic reactions often involve the attack by lone pairs of
electrons residing on electron-rich molecules termed nucleo-
philes upon electron-poor atoms called electrophiles. Nucleo-
philes and electrophiles do not necessarily possess a formal
negative or positive charge. Water, whose two lone pairs of
sp3 electrons bear a partial negative charge (see Figure 2–1),
is an excellent nucleophile. Other nucleophiles of biologic
importance include the oxygen atoms of phosphates, alcohols,
and carboxylic acids; the sulfur of thiols; and the nitrogen
atoms of amines and of the imidazole ring of histidine. Com-
mon electrophiles include the carbonyl carbons in amides,
esters, aldehydes, and ketones and the phosphorus atoms of
phosphoesters.
Nucleophilic attack by water typically results in the cleav-
age of the amide, glycoside, or ester bonds that hold biopoly-
mers together. This process is termed hydrolysis. Conversely,
when monomer units such as amino acids or monosaccha-
rides are joined or condensed together to form biopolymers,
such as proteins or starch, water is a product.
Hydrolysis typically is a thermodynamically favored reac-
tion. Yet, the amide and phosphoester bonds of polypeptides
CHAPTER 2 Water & pH 9
and oligonucleotides are stable in the aqueous environment of
the cell. This seemingly paradoxical behavior reflects the fact
that the thermodynamics that govern the equilibrium point
of a reaction do not determine the rate at which it will pro-
catalysts called enzymes accelerate the rate of hydrolytic and
other chemical reactions when needed. Proteases catalyze
the hydrolysis of proteins into their component amino acids,
while nucleases catalyze the hydrolysis of the phosphoester
bonds in DNA and RNA. Precise and differential control of
6.0 7.0 8.0
enzyme activity, including the sequestration of enzymes in
specific organelles, enables cells to determine the physiologic
circumstances under which a given biopolymer will be synthe-
sized or degraded.
Many Metabolic Reactions Involve
Group Transfer
Many of the enzymic reactions responsible for synthesis and
breakdown of biomolecules involve the transfer of a chemical
group G from a donor D to an acceptor A to form an acceptor
group complex, A—G:
D—G + A  A—G + D
The hydrolysis and phosphorolysis of glycogen, for example,
involve the transfer of glucosyl groups to water or to ortho-
phosphate. Since the equilibrium constants for these hydro-
lysis reactions strongly favor the formation of split products,
it follows that many of the group transfer reactions respon-
sible for the biosynthesis of macromolecules are, in and of
themselves, thermodynamically unfavored. Enzyme catalysts
play a critical role in surmounting these barriers by virtue of
their capacity to directly link two normally separate reactions
together. For example, by linking an energetically unfavorable
group transfer reaction to a thermodynamically favorable one
such as the hydrolysis of ATP, a new enzyme-catalyzed reac-
tion can be generated. The free energy change of this coupled
reaction will be the sum of the individual values for the two
that were linked, one whose net overall change in free energy
favors the formation of the covalent bonds required for bio-
polymer synthesis.
Water Molecules Exhibit a Slight but
Important Tendency to Dissociate
The ability of water to ionize, while slight, is of central importance
for life. Since water can act both as an acid and as a base, its ioniza-
tion may be represented as an intermolecular proton transfer that
forms a hydronium ion (H3O+) and a hydroxide ion (OH−):
H2 O + H2 O  H3O + OH−
The transferred proton is actually associated with a cluster of
water molecules. Protons exist in solution not only as H3O+
but also as multimers such as H5O2+ and H7O3+. The proton is
nevertheless routinely represented as H+, even though it is in
fact highly hydrated.
10 SECTION I Structures & Functions of Proteins & Enzymes
Since hydronium and hydroxide ions continuously recom-
bine to form water molecules, an individual hydrogen or oxy-
gen cannot be stated to be present as an ion or as part of a water
molecule. At one instant it is an ion; an instant later it is part
of a water molecule. Individual ions or molecules are therefore
not considered. We refer instead to the probability that at any
instant in time, a given hydrogen will be present as an ion or
as part of a water molecule. Since 1 g of water contains 3.35 ×
1022 molecules, the ionization of water can be described statis-
tically. To state that the probability that a hydrogen exists as an
ion is 0.01 means that at any given moment in time, a hydro-
gen atom has 1 chance in 100 of being an ion and 99 chances
out of 100 of being part of a water molecule. The actual prob-
ability of a hydrogen atom in pure water existing as a hydrogen
ion is approximately 1.8 × 10−9. The probability of its being
part of a water molecule thus is almost unity. Stated another
way, for every hydrogen ion or hydroxide ion in pure water,
there are 0.56 billion or 0.56 × 109 water molecules. Hydrogen
ions and hydroxide ions nevertheless contribute significantly
to the properties of water.
For dissociation of water,
+
[H ][OH ] − ical purposes. To calculate the pH of a solution:
K=
[H2 O]
where the brackets represent molar concentrations (strictly
speaking, molar activities) and K is the dissociation constant.
Since 1 mole (mol) of water weighs 18 g, 1 liter (L) (1000 g)
of water contains 1000 ÷ 18 = 55.56 mol. Pure water thus is
55.56 molar. Since the probability that a hydrogen in pure
water will exist as a hydrogen ion is 1.8 × 10−9, the molar con-
centration of H+ ions (or of OH− ions) in pure water is the
product of the probability, 1.8 × 10−9, times the molar concen-
tration of water, 55.56 mol/L. The result is 1.0 × 10−7 mol/L.
We can now calculate the dissociation constant K for pure
water:
[H+ ][OH− ] [10−7 ][10−7 ]
K= =
[H2 O] [55.56]
= 0.018 × 10−14 = 1.8 ×10−16 mol/L
The molar concentration of water, 55.56 mol/L, is too great
to be significantly affected by dissociation. It is therefore con-
sidered to be essentially constant. The concentration of pure
water may therefore be incorporated into the dissociation con-
stant K to provide a useful new constant Kw termed the ion
product for water:
[H+ ][OH− ]
K= = 1.8 × 10−16 mol/L
[H2O]
K w = ( K )[H2O] = [H+ ][OH− ]
−16
= (1.8 × 10 mol/L)(55.56mol/L)
−14 2
= 1.00 × 10 (mol/L)
Note that the dimensions of K are moles per liter and those of
Kw are moles2 per liter2. As its name suggests, the ion product
Kw is numerically equal to the product of the molar concentra-
tions of H+ and OH−:
K w = [H+ ][OH− ]
At 25°C, Kw = (10−7)2, or 10−14 (mol/L)2. At temperatures below
25°C, Kw is somewhat less than 10−14, and at temperatures above
25°C it is somewhat greater than 10−14. Within the stated limi-
tations of temperature, Kw equals 10−14 (mol/L)2 for all aqueous
solutions, even solutions containing acids or bases. We can
therefore use Kw to calculate the pH of any aqueous solution.
pH IS THE NEGATIVE LOG OF THE
HYDROGEN ION CONCENTRATION
The term pH was introduced in 1909 by Sörensen, who defined
it as the negative log of the hydrogen ion concentration:
pH = − log[H+ ]
This definition, while not rigorous, suffices for most biochem-
1. Calculate the hydrogen ion concentration [H+].
2. Calculate the base 10 logarithm of [H+].
3. pH is the negative of the value found in step 2.
For example, for pure water at 25°C,
pH = − log[H+ ] = − log10−7 = −(−7) = 7.0
This value is also known as the power (English), puissant
(French), or potennz (German) of the exponent, hence the use
of the term “p.”
Low pH values correspond to high concentrations of H+
and high pH values correspond to low concentrations of H+.
Acids are proton donors and bases are proton acceptors.
Strong acids (eg, HCl, H2SO4) completely dissociate into anions
and protons even in strongly acidic solutions (low pH). Weak
acids dissociate only partially in acidic solutions. Similarly, strong
bases (eg, KOH, NaOH), but not weak bases like Ca(OH)2, are
completely dissociated even at high pH. Many biochemicals are
weak acids. Exceptions include phosphorylated intermediates,
whose phosphoryl group contains two dissociable protons, the
first of which is strongly acidic.
The following examples illustrate how to calculate the pH
of acidic and basic solutions.
Example 1: What is the pH of a solution whose hydrogen
ion concentration is 3.2 × 10−4 mol/L?
pH = − log[H+ ]
= − log(3.2 × 10−4 )
= − log(3.2) − log(10−4 )
= − 0.5 + 4.0
= 3.5
 CHAPTER 2 Water & pH 11

Example 2: What is the pH of a solution whose hydroxide that present initially in the water. This assumption is valid
ion concentration is 4.0 × 10−4 mol/L? We first define a quan- for dilute solutions of strong bases or acids, but not for weak
tity pOH that is equal to −log[OH−] and that may be derived bases or acids. Since weak electrolytes dissociate only slightly
from the definition of Kw: in solution, we must use the dissociation constant to calcu-
late the concentration of [H+] (or [OH−]) produced by a given
K w = [H+ ][OH− ] = 10−14 molarity of a weak acid (or base) before calculating total [H+]
(or total [OH−]) and subsequently pH.
Therefore,
log[H+ ] + log[OH− ] = log10−14 Functional Groups That Are Weak Acids
Have Great Physiologic Significance
or
Many biomolecules contain functional groups that are weak
pH + pOH = 14
acids or bases. Carboxyl groups, amino groups, and phosphate
To solve the problem by this approach: esters, whose second dissociation falls within the physiologic
range, are present in proteins and nucleic acids, most coen-
[OH− ] = 4.0 × 10−4 zymes, and most intermediary metabolites. Knowledge of the
dissociation of weak acids and bases thus is basic to under-
pOH = − log[OH− ]
standing the influence of intracellular pH on structure and bio-
= − log(4.0 × 10−4 ) logic activity. Charge-based separations such as electrophoresis
= − log(4.0) − log(10−4 ) and ion exchange chromatography are also best understood in
terms of the dissociation behavior of functional groups.
= −0.60 + 4.0
When discussing weak acids, we often refer to the proton-
= 3.4 ated species (HA or R—SH) as the acid and the unprotonated
species (A− or R—S−) as its conjugate base. Similarly, we may
Now
refer to the deprotonated form as the base (A− or R—COO−) and
pH = 14 − pOH = 14 − 3.4 the protonated form as its conjugate acid (HA or R—COOH).
= 10.6 We express the relative strengths of weak acids in terms
of the dissociation constants of the protonated form. Follow-
Examples 1 and 2 illustrate how the logarithmic pH scale facili- ing are the expressions for the dissociation constant (Ka) for a
tates recording and comparing hydrogen ion concentrations representative weak acid, R—COOH, as well as the conjugate
that differ by orders of magnitude from one another, 0.00032 M acid, R—NH3+, of the weak base R—NH2.
(pH 3.5) and 0.000000000025 M (pH 10.6).
Example 3: What are the pH values of (a) 2.0 × 10−2 mol/L R—COOH  R—COO− + H+
KOH and of (b) 2.0 × 10−6 mol/L KOH? The OH− arises from [R —COO− ][H+ ]
two sources, KOH and water. Since pH is determined by the Ka =
[R—COOH]
total [H+] (and pOH by the total [OH−]), both sources must be
considered. In the first case (a), the contribution of water to R—NH3+  R—NH2 + H+
the total [OH−] is negligible. The same cannot be said for the [R—NH2 ][H+ ]
second case (b): Ka =
[R—NH3+ ]

Concentration (mol/L)
Since the numeric values of Ka for weak acids are negative
(a) (b) exponential numbers, we express Ka as pKa, where
Molarity of KOH 2.0 × 10−2 2.0 × 10−6 pK a = − log K a
− −2 −6
[OH ] from KOH 2.0 × 10 2.0 × 10
Note that pKa is related to Ka as pH is to [H+]. The stronger the
− −7
[OH ] from water 1.0 × 10 1.0 × 10−7 acid, the lower is its pKa value.
Total [OH−] 2.00001 × 10−2 2.1 × 10−6 Representative weak acids (left), their conjugate bases
(center), and pKa values (right) include the following:

Once a decision has been reached about the significance of R—CH2 —COOH R—CH2 COO− pK a = 4 − 5
the contribution of water, pH may be calculated as shown in
Example 3. R—CH2 —NH3+ R—CH2 —NH2 pK a = 9 − 10
The above examples assume that the strong base KOH H2 CO3 HCO3 −
pK a = 6.4
is completely dissociated in solution and that the concentra- − −2
tion of OH− ions was thus equal to that due to the KOH plus H2 PO4 HPO4 pK a = 7.2
12 SECTION I Structures & Functions of Proteins & Enzymes

pKa is used to express the relative strengths of both weak Cross-multiplication gives
acids and weak bases using a single, unified scale. Under this
convention, the relative strengths of bases are expressed [H+ ][A− ] = K a [HA]
in terms of the pKa of their conjugate acids. For polyprotic
Divide both sides by [A−]:
compounds containing more than one dissociable proton, a
numerical subscript is assigned to each dissociation, numbered [HA]
starting from unity in decreasing order of relative acidity. For a [H+ ] = K a
[A − ]
dissociation of the type
R—NH3+ → R—NH 2 + H + Take the log of both sides:

the pKa is the pH at which the concentration of the acid  [HA] 

log[H+ ] = log  K a − 
R—NH3+ equals that of the base R—NH2.  [A ] 
From the above equations that relate Ka to [H+] and to the [HA]
concentrations of undissociated acid and its conjugate base, when = log K a + log −
[A ]
[R—COO− ] = [R—COOH]
Multiply through by −1:
or when
[HA]
− log[H+ ] = − log K a − log
[R—NH2 ] = [R—NH3+ ] [A− ]

then Substitute pH and pKa for −log [H+] and −log Ka, respectively;
K a = [H ]+ then
[HA]
Thus, when the associated (protonated) and dissociated pH = pK a − log
[A− ]
(conjugate base) species are present at equal concentrations,
the prevailing hydrogen ion concentration [H+] is numerically Inversion of the last term removes the minus sign and gives
equal to the dissociation constant, Ka. If the logarithms of both the Henderson-Hasselbalch equation
sides of the above equation are taken and both sides are multi-
plied by −1, the expressions would be as follows: [A − ]
pH = pK a + log
K a = [H+ ] [HA]

− log K a = − log[H+ ] The Henderson-Hasselbalch equation has great predictive

value in protonic equilibria. For example,
Since −log Ka is defined as pKa, and −log [H+] defines pH, the
equation may be rewritten as 1. When an acid is exactly half-neutralized, [A−] = [HA].
Under these conditions,
pK a = pH
[A − ] 1 
that is, the pKa of an acid group is the pH at which the pH = pK a + log = pK a + log   = pK a + 0
[HA] 1 
protonated and unprotonated species are present at equal
concentrations. The pKa for an acid may be determined by Therefore, at half-neutralization, pH = pKa.
adding 0.5 equivalent of alkali per equivalent of acid. The
resulting pH will equal the pKa of the acid. 2. When the ratio [A−]/[HA] = 100:1,

[A − ]
The Henderson-Hasselbalch Equation pH = pK a + log
[HA]
Describes the Behavior of Weak Acids & pH = pK a + log(100/1) = pK a + 2
Buffers
The Henderson-Hasselbalch equation is derived below.
3. When the ratio [A−]/[HA] = 1:10,
A weak acid, HA, ionizes as follows:
pH = pK a + log(1/10) = pK a + (−1)
HA  H+ + A−

The equilibrium constant for this dissociation is If the equation is evaluated at ratios of [A−]/[HA] ranging
from 103 to 10−3 and the calculated pH values are plotted, the
[H+ ][A− ] resulting graph describes the titration curve for a weak acid
Ka =
[HA] (Figure 2–6).
 CHAPTER 2 Water & pH 13

1.0 –1.0 Notice that ΔpH, the change in pH per milliequivalent of

meq of alkali added per meq of acid

OH− added, depends on the initial pH, with highest resistance
0.8 –0.8
to change at pH values close to the weak acid’s pKa. Indeed,
such weak acid-conjugate base combinations, called buf-

Net charge
0.6 –0.6
fers, resist change most effectively when the desired pH falls
0.4 –0.4 within, ± 1.0 unit or less of their pKa.
Figure 2–6 also illustrates how the net charge on one
0.2 –0.2 molecule of a weak acid varies with pH. A fractional charge
of −0.5 does not mean that an individual molecule bears a
0 0
2 3 4 5 6 7 8
fractional charge but that the probability is 0.5 that a given
pH
molecule has a unit negative charge at any given moment in
time. Consideration of the net charge on macromolecules as
FIGURE 2–6 Titration curve for an acid of the type HA. The a function of pH provides the basis for separatory techniques
heavy dot in the center of the curve indicates the pKa, 5.0. such as ion exchange chromatography and electrophoresis
(see Chapter 4).
Weak Acids Can Be Used to Establish &
Maintain the pH of an Aqueous Solution The Propensity of a Proton to
Solutions of weak acids or bases and their conjugates exhibit Dissociate Depends on Molecular
buffering, the ability to resist a change in pH following addi- Structure
tion of strong acid or base. Many metabolic reactions are Many acids of biologic interest possess more than one dissoci-
accompanied by the release or uptake of protons. Oxidative ating group. The presence of local negative charge hinders pro-
metabolism produces CO2, the anhydride of carbonic acid, ton release from nearby acidic groups, raising their pKa. This
which if not buffered would produce severe acidosis. Bio- is illustrated by the pKa values of the three dissociating groups
logic maintenance of a constant pH involves buffering by of phosphoric acid and citric acid (Table 2–2). The effect of
phosphate, bicarbonate, and proteins, which accept or release adjacent charge decreases with distance. The second pKa for
protons to resist a change in pH. For laboratory experiments succinic acid, which has two methylene groups between its
using tissue extracts or enzymes, constant pH is maintained carboxyl groups, is 5.6, whereas the second pKa for glutaric
by the addition of buffers such as MES ([2-N-morpholino]- acid, which has one additional methylene group, is 5.4.
ethanesulfonic acid, pKa 6.1), inorganic orthophosphate (pKa2 7.2),
HEPES (N-hydroxyethylpiperazine-N′-2-ethanesulfonic acid,
pKa 6.8), or Tris (tris[hydroxymethyl]aminomethane, pKa 8.3). pKa Values Depend on the
The value of pKa relative to the desired pH is the major deter- Properties of the Medium
minant of which buffer is selected. The pKa of a functional group is also profoundly influenced
Buffering can be observed by using a pH meter while titrat- by the surrounding medium. The medium may either raise
ing a weak acid or base (see Figure 2–6). We can also calculate or lower the pKa relative to its value in water, depending on
the pH shift that accompanies addition of acid or base to a buff- whether the undissociated acid or its conjugate base is the
ered solution. In the following example, the buffered solution charged species. The effect of dielectric constant on pKa may
(a weak acid, pKa = 5.0, and its conjugate base) is initially at one be observed by adding ethanol to water. The pKa of a carbox-
of four pH values. We will calculate the pH shift that results ylic acid increases, whereas that of an amine decreases on addi-
when 0.1 meq of KOH is added to 1 meq of each solution: tion of ethanol because ethanol decreases the ability of water
to solvate a charged species. The pKa values of dissociating
Initial pH 5.00 5.37 5.60 5.86
−
TABLE 2−2 Relative Strengths of Monoprotic, Diprotic,
[A ]initial 0.50 0.70 0.80 0.88
and Triprotic Acids
[HA]initial 0.50 0.30 0.20 0.12
Lactic acid pK = 3.86
−
([A ]/[HA])initial 1.00 2.33 4.00 7.33
Acetic acid pK = 4.76
Addition of 0.1 meq of KOH Produces
Ammonium ion pK = 9.25
[A−]final 0.60 0.80 0.90 0.98
Carbonic acid pK1 = 6.37; pK2 = 10.25
[HA]final 0.40 0.20 0.10 0.02
Succinic acid pK1 = 4.21; pK2 = 5.64
([A−]/[HA])final 1.50 4.00 9.00 49.0
Glutaric acid pK1 = 4.34; pK2 = 5.41
log ([A−]/[HA])final 0.18 0.60 0.95 1.69
Phosphoric acid pK1 = 2.15; pK2 = 6.82; pK3 = 12.38
Final pH 5.18 5.60 5.95 6.69
Citric acid pK1 = 3.08; pK2 = 4.74; pK3 = 5.40
ΔpH 0.18 0.60 0.95 1.69
Note: Tabulated values are the pKa values (-log of the dissociation constant).
14 SECTION I Structures & Functions of Proteins & Enzymes
groups in the interiors of proteins thus are profoundly affected
by their local environment, including the presence or absence
of water.
SUMMARY
■ Water forms hydrogen-bonded clusters with itself and with
other proton donors or acceptors. These extensive networks
of hydrogen bonds account for the surface tension, viscosity,
liquid state at room temperature, and solvent power of water.
■ Compounds that contain O or N can serve as hydrogen bond
donors and/or acceptors.
■ Entropic forces dictate that amphipathic macromolecules bury
nonpolar regions away from water.
■ Salt bridges, hydrophobic interactions, and van der Waals
forces participate in the formation of biomolecular complexes
and maintenance of molecular conformation.
■ pH is the negative log of [H+]. A low pH characterizes an acidic
solution, and a high pH denotes a basic solution.
■ The strength of weak acids is expressed by pKa, the negative
log of the acid dissociation constant. Strong acids have low pKa
values and weak acids have high pKa values.
■ Buffers resist a change in pH when protons are produced
or consumed. Maximum buffering capacity occurs within
1 pH unit on either side of pKa. Physiologic buffers include
bicarbonate, orthophosphate, and proteins.
REFERENCES
Reese KM: Whence came the symbol pH. Chem & Eng News
2004;82:64.
Segel IM: Biochemical Calculations. Wiley, 1968.
Skinner JL: Following the motions of water molecules in aqueous
solutions. Science 2010;328:985.
Stillinger FH: Water revisited. Science 1980;209:451.
Suresh SJ, Naik VM: Hydrogen bond thermodynamic properties of
water from dielectric constant data. J Chem Phys 2000;113:9727.
Wiggins PM: Role of water in some biological processes. Microbiol
Rev 1990;54:432.
 C H A P T E R

Amino Acids & Peptides

Peter J. Kennelly, PhD, & Victor W. Rodwell, PhD
OBJ E C TI VE S
After studying this chapter,
you should be able to:
3
■ Diagram the structures and write the three- and one-letter designations for
each of the amino acids present in proteins.
■ Provide examples of how each type of R group of the protein amino acids
contributes to their chemical properties.
■ List additional important functions of amino acids and explain how certain
amino acids in plant seeds can severely impact human health.
■ Name the ionizable groups of the protein amino acids and list their
approximate pKa values as free amino acids in aqueous solution.
■ Calculate the pH of an unbuffered aqueous solution of a polyfunctional
amino acid and the change in pH that occurs following the addition of a given
quantity of strong acid or base.
■ Define pI and explain its relationship to the net charge on a polyfunctional
electrolyte.
■ Explain how pKa and pI can be used to predict the mobility at a given pH of a
polyelectrolyte, such as an amino acid, in a direct-current electrical field.
■ Describe the directionality, nomenclature, and primary structure of peptides.
■ Describe the conformational consequences of the partial double-bond
character of the peptide bond.

BIOMEDICAL IMPORTANCE
l-α-Amino acis provie the monomer units of the ong poy-
peptie chains of proteins. In aition, these amino acis an
their erivatives participate in such iverse ceuar functions
as nerve transmission an the biosynthesis of porphyrins,
purines, pyrimiines, an urea. The neuroenocrine system
empoys short poymers of amino acis cae peptides as
hormones, hormone-reeasing factors, neuromouators, an
neurotransmitters. Humans an other higher animas can-
not synthesize 10 of the l-α-amino acis present in proteins
in amounts aequate to support infant growth or to maintain
aut heath. Consequenty, the human iet must contain ae-
quate quantities of these nutritionally essential amino acis.
Each ay the kineys fiter over 50 g of free amino acis from
the arteria rena boo. However, ony traces of free amino
acis normay appear in the urine because amino acis are
amost totay reabsorbe in the proxima tubue, conserving
them for protein synthesis an other vita functions.
Certain microorganisms secrete free d-amino acis, or
pepties that may contain both d- an l-α-amino acis. Severa
of these bacteria pepties are of therapeutic vaue, incuing
the antibiotics bacitracin an gramiciin A, an the antitu-
mor agent beomycin. Certain other microbia pepties are,
however, toxic. The cyanobacteria pepties microcystin an
nouarin are etha in arge oses, whie sma quantities pro-
mote the formation of hepatic tumors. The ingestion of cer-
tain amino acis present in the sees of egumes of the genus
Lathyrus can resut in athyrism, a tragic irreversibe isease
in which iniviuas ose contro of their imbs. Certain other
pant see amino acis have aso been impicate in a neuro-
egenerative isease afficting natives of Guam.

15
16 SECTION I Structures & Functions of Proteins & Enzymes

PROPERTIES OF AMINO ACIDS
The Genetic Code Specifies
20 l-α-Amino Acids
Athough more than 300 amino acis occur in nature, pro-
teins are synthesize amost excusivey from the set of 20 l-α-
amino acis encoe by nuceotie tripets cae codons (see
Tabe 37–1). Whie the three-etter genetic coe cou poten-
tiay accommoate more than 20 amino acis, the genetic coe
is redundant since severa amino acis are specifie by mutipe
coons. Scientists frequenty represent the sequences of pepties
an proteins using one- an three-etter abbreviations for each
amino aci (Table 3–1). The R groups of amino acis may be
either hyrophiic or hyrophobic (Table 3–2); properties that
affect their ocation in a protein’s mature foe conformation
(see Chapter 5). Some proteins contain aitiona amino acis
that arise by the posttranslational moification of an amino
aci areay present in a peptie. Exampes incue the conver-
sion of peptiy proine an peptiy ysine to 4-hyroxyproine
an 5-hyroxyysine; the conversion of peptiy gutamate to
γ-carboxygutamate; an the methyation, formyation, acety-
ation, prenyation, an phosphoryation of certain aminoacy
resiues. These moifications significanty exten the func-
tiona iversity of proteins by atering their soubiity, stabiity,
cataytic activity, an interaction with other proteins.

TABLE 3−1 l-α-Amino Acids Present in Proteins

Name Symbol Structural Formula pK1 pK2 pK3

With Aliphatic Side Chains α-COOH α-NH2+ R Group

Glycine Gly[G] 2.4 9.8

Alanine Ala[A] 2.4 9.9

Valine Val[V] 2.2 9.7

Leucine Leu[L] 2.3 9.7

Isoleucine Ile[T] 2.3 9.8

With Side Chains Containing Hydroxylic(OH) Groups

Serine Ser[S] 2.2 9.2 about 13

Threonine Thr[T] 2.1 9.1 about 13

Tyrosine Tyr[Y] See below.

With Side Chains Containing Sulfur Atoms

Cysteine Cys[C] 1.9 10.8 8.3

(Continued)
 CHAPTER 3 Amino Acids & Peptides 17

TABLE 3−1 l-α-Amino Acids Present in Proteins (Continued)

Name Symbol Structural Formula pK1 pK2 pK3

With Side Chains Containing Sulfur Atoms

Methionine Met[M] 2.1 9.3

With Side Chains Containing Acidic Groups or Their Amides

Aspartic Acid Asp[D] 2.1 9.9 3.9

Asparagine Asn[N] 2.1 8.8

Glutamic Acide Glu[E] 2.1 9.5 4.1

Glutamine Gin[Q] 2.2 9.1

With Side Chains Containing Basic Groups

Argine Arg[R] 1.8 9.0 12.5

Lysine Lys[K] 2.2 9.2 10.8

Histidine His[H] 1.8 9.3 6.0

Containing Aromatic Rings

Histidine His[H] See above
Phenylalanine Phe[F] 2.2 9.2

Tyrosine Try[Y] 2.2 9.1 10.1

Tryptophan Trp[W] 2.4 9.4

Imino Acid
Proline Pro[P] 2.0 10.6
18 SECTION I Structures & Functions of Proteins & Enzymes

TABLE 3−2 Hydrophilic & Hydrophobic Amino Acids HO

OH

Hydrophilic Hydrophobic H2N COOH

N COOH
Arginine Alanine H

Asparagine Isoleucine NH2

Aspartic acid Leucine FIGURE 3–2 4-Hydroxyproline and 5-hydroxylysine.

Cysteine Methionine
configuration of l-gyceraehye an thus are efine as l-α-
Glutamic acid Phenylalanine amino acis. Even though amost a protein amino acis are
Glutamine Proline (S), the faiure to use (R) or (S) to express absolute stereochem-
Glycine Tryptophan
istry is no mere historica aberration. l-Cysteine is (R) since
the atomic mass of the sufur atom on C3 excees that of the
Histidine Tyrosine amino group on C2. More significanty, in mammas the bio-
Lysine Valine chemica reactions of l-α-amino acis, their precursors, an
Serine
their cataboites are catayze by enzymes that act excusivey
on l-isomers, irrespective of their absoute configuration.
Threonine

The distinction is based on the tendency of their R groups to associate with, or
to minimize contact with, an aqueous environment.
Selenocysteine, the 21st
Protein l-α-Amino Acid
Seenocysteine (Figure 3–1) is an l-α-amino aci present in pro-
teins from every omain of ife. Humans contain approximatey
two ozen seenoproteins that incue certain peroxiases an
reuctases, seenoprotein P, which circuates in the pasma,
an the ioothyronine eioinases responsibe for converting
the prohormone thyroxine (T4) to the thyroi hormone 3,3′,
5-triioothyronine (T3) (see Chapter 41). Since peptiy seeno-
cysteine is inserte irecty into a growing poypeptie uring
translation, it is commony terme the “21st amino aci.” How-
ever, unike the other 20 protein amino acis, incorporation of
seenocysteine is specifie by a arge an compex genetic eement
for the unusua tRNA cae tRNASec which utiizes the UGA anti-
coon that normay signas STOP rather than a simpe tripet
coon. Rather, the protein synthetic apparatus recognizes a UGA
coon as coing for seenocysteine when it is accompanie by a
stem-oop structure, the seenocysteine insertion eement, in the
untransate region of the mRNA (see Chapter 27).
Posttranslational Modifications
Confer Additional Properties
Whie some prokaryotes incorporate pyrroysine into pro-
teins, an pants can incorporate azetiine-2-carboxyic aci,
an anaog of proine, a set of just 21 l-α-amino acis ceary
suffices for the formation of most proteins. Posttransationa
moifications can, however, generate nove R groups that
impart further properties. In coagen, protein-boun pro-
ine an ysine resiues are converte to 4-hyroxyproine
an 5-hyroxyysine (Figure 3–2). The carboxyation of gu-
tamy resiues of proteins of the boo coaguation cascae to
γ-carboxygutamy resiues (Figure 3–3) competes a site for
cheating the cacium ion essentia for boo coaguation. The
amino aci sie chains of histones are subject to numerous
moifications, incuing acetyation an methyation of ysine
an methyation an eimination of arginine (see Chapters 35
an 38). It is aso now possibe in the aboratory to geneticay
introuce many ifferent unnatura amino acis into proteins,
generating proteins via recombinant gene expression with new
or enhance properties an proviing a new way to expore
protein structure–function reationships.

Stereochemistry of the Protein Extraterrestrial Amino Acids Have Been

Amino Acids Detected in Meteorites
The existence of extraterrestria amino acis was first reporte
With the soe exception of gycine, the α-carbon of every
in 1969 foowing anaysis of the famous Murchison meteor-
amino aci is chira. Athough some protein amino acis are
ite from southeastern Austraia. The presence of amino acis
extrorotatory an some evorotatory, a share the absoute
in other meteorites, incuing some pristine exampes from
NH3
+
NH3
+ Antarctica, has now been ampy confirme. Unike the amino
acis synthesize by terrestria organisms, these meteorites
O– O–
HS HSe COOH

O O
HOOC
FIGURE 3–1 Cysteine (left) and selenocysteine (right). pK3,
for the selenyl proton of selenocysteine is 5.2. Since this is 3 pH H2N COOH
units lower than that of cysteine, selenocysteine represents a better
nucleophile at or below pH 7.4. FIGURE 3–3 γ-Carboxyglutamic acid.
 CHAPTER 3 Amino Acids & Peptides 19

contain racemic mixtures of d- an l-isomers of mutipe TABLE 3−3 Potentially Toxic l-α-Amino Acids
protein amino acis as we as bioogicay important nonpro-
Nonprotein l-α-Amino Acid Medical Relevance
tein α-amino acis such as N-methygycine (sarcosine) an
β-aanine. Severa nove amino acis that ack terrestria coun- NH NH2 Cleaved by arginase
terparts of biotic origin were aso iscovere. Nuceobases, acti- to l-lysine and urea.
OH
H2N N Implicated in human
vate phosphates, an moecues reate to sugars have aso been H neurolathyrism.
O
etecte in meteorites. These finings offer potentia insights Homoarginine
into the prebiotic chemistry of earth, an impact the search for
extraterrestria ife. Some specuate that meteorites may have O NH2 A neurotoxin.
H
N Implicated in human
contribute to the origin of ife on our panet, a conjecture OH
neurolathyrism.
HO
entite Panspermia, by eivering extraterrestriay generate
O O
organic moecues or even intact microorganisms to our earth. -N-Oxalyl
diaminopropionic acid ( -ODAP)
l-α-Amino Acids Serve Additional An osteolathyrogen.
O NH2
Metabolic Roles OH
H2N
l-α-Amino acis fufi vita metaboic roes in aition to
NH O
serving as the “buiing bocks” of proteins. For exampe, orni-
N
thine an citruine are key intermeiates in the urea cyce H3C C
(see Figure 28–16), whie S-aenosy-methionine serves as a -N-Glutamylamino-propiononitrile
methy-group onor for many enzyme-catayze reactions. (BAPN)
Tyrosine is a precursor of thyroi hormone, whie both tyro- Inhibits ornithine
NH2 NH2
sine an phenyaanine are metaboize to prouce epineph- transcarbamylase,
OH
rine, norepinephrine, an ihyroxyphenyaanine (DOPA). resulting in ammonia
Gutamate is both a neurotransmitter as we as a precursor of a O toxicity.
secon neurotransmitter, γ-aminobutyric aci (GABA). 2,4-Diaminobutyric acid

CH3 Possible risk factor for

Certain Plant l-α-Amino Acids Can HN
NH
neurodegenerative
diseases.
Adversely Impact Human Health OH

The consumption of pants that contain certain nonprotein O

amino acis can aversey impact human heath. The sees an
see proucts of three species of the egume Lathyrus have been
impicate in the genesis of neurolathyrism, a profoun neuro-
ogic isorer characterize by progressive an irreversibe spas-
tic paraysis of the egs. Lathyrism occurs wiey uring famines,
when Lathyrus sees may become a major part of the iet. l-α-
Amino acis that have been impicate in human neuroogic is-
orers, notaby neuroathyrisms, incue l-homoarginine an
β-N-oxay-l-α,β-iaminopropionic aci (β-ODAP Table 3–3).
The sees of another Lathyrus egume, the “sweet pea,” contain
the osteoathyrogen γ-gutamy-β-aminopropionitrie (BAPN),
a gutamine erivative of β-aminopropionitrie (structure not
shown). The sees of certain Lathyrus species aso contain α,γ-
iaminobutyric aci, an anaog of ornithine that inhibits the
hepatic urea cyce enzyme ornithine transcarbamyase, eaing
to ammonia toxicity. Finay, l-β-methyaminoaanine, a neuro-
toxic amino aci that is present in Cycad sees, has been impi-
cate as a risk factor for neuroegenerative iseases incuing
amyotrophic atera scerosis–Parkinson ementia compex in
natives of Guam who consume either fruit bats that fee on
cyca fruit, or four mae from cyca sees.
d-Amino Acids
d-Amino acis occur naturay throughout the biosphere,
incuing free d-serine an d-aspartate in human brain tissue,
-Methylaminoalanine
d-aanine an d-gutamate in the ce was of gram-positive
bacteria, an d-amino acis in certain pepties an antibiotics
prouce by bacteria, fungi, repties, an amphibians. Bacil-
lus subtilis excretes d-methionine, d-tyrosine, d-eucine, an
d-tryptophan to trigger biofim isassemby, an Vibrio chol-
erae incorporates d-eucine an d-methionine into the pep-
tie component of its peptiogycan ayer.
PROPERTIES OF THE FUNCTIONAL
GROUPS OF AMINO ACIDS
Amino Acids May Have Positive,
Negative, or Zero Net Charge
In aqueous soution, the charge an uncharge forms of the
ionizabe weak aci groups —COOH an —NH3+ exist in
ynamic protonic equiibrium:
R—COOH ⇄ R—COO– + H+
R—NH3+ ⇄ R—NH2 + H+
Whie both R—COOH an R—NH3+ are weak acis, R—COOH
is a far stronger aci than R—NH3+. Thus, at physioogic pH
20 SECTION I Structures & Functions of Proteins & Enzymes
R R
N H
N N
H H
R R
NH NH
C NH2 C NH2
NH2 NH2
FIGURE 3–4 The protonated R groups of histidine (top) and
arginine (bottom) are stabilized by electronic resonance.
(pH 7.4), the α-carboxy groups of free amino acis exist
amost entirey as R—COO− an α-amino groups preomi-
nanty as R—NH3+. The same is true for the sie chain car-
boxy groups of aspartic aci an gutamic aci as we as the
ε-amino group of ysine. Other ionizabe groups incue the
sufhyry group of cysteine, the imiazoe ring of histiine
an the guaniino group of arginine (Figure 3–4). Figure 3–5
iustrates the effect that the pH of the aqueous environment
has on the charge state of aspartic aci.
Amino acis in boo an most tissues thus shou be rep-
resente as in A, in the foowing figure. Note that these ion-
ize yet net neutra moecues, a consequence of their equa
numbers of positivey an negativey charge groups, are
terme zwitterions.
NH3+ NH2
O–
R R
O O
A B
Structure B cannot exist in aqueous soution because at
any pH ow enough to protonate the carboxy group, the amino
group wou aso be protonate. Simiary, at any pH suffi-
cienty high for an uncharge amino group to preominate,
a carboxy group wi be present as R—COO−. The uncharge
representation B is, however, often use when iagramming
reactions that o not invove protonic equiibria.
O H+
OH
pK1 = 2.09
NH3+ (α-COOH)
HO
O O
A B
In strong acid
(below pH 1);
net charge = +1
FIGURE 3–5 Protonic equilibria of aspartic acid.
pKa Values Express the Strengths
N H of Weak Acids & Bases
The strengths of weak acis are expresse as their pKa. For
moecues with mutipe issociabe protons, the pKa for each
aciic group is esignate by repacing the subscript “a” with
R
a number. The strength of weak bases is generay expresse
as the pKa of their protonate, or conjugate aci, form. This
NH
enabes the reative strengths of a groups capabe of issocia-
C NH2 by bining proteins to be compare on a singe, continuous
NH2 scae. The terms “weak aci” an “weak base” thus consti-
tute arbitrary abes since in any protonic equiibrium both
aciic an basic forms must participate. The net charge on an
amino aci—the agebraic sum of a the positivey an nega-
tivey charge groups present—epens on the pKa vaues of
its functiona groups an the pH of the surrouning meium.
In the aboratory, atering the charge on amino acis an their
erivatives by varying the pH faciitates the physica separation
of amino acis, pepties, an proteins (see Chapter 4).
At Its Isoelectric pH (pI), an Amino Acid
Bears No Net Charge
Zwitterions are one exampe of an isoelectric species—the
form of a moecue that has an equa number of positive an
negative charges an thus is eectricay neutra. The isoeec-
tric pH, aso cae the isoeectric point or pI, is the pH mi-
way between pKa vaues for the ionizations on either sie of
the isoeectric species. For an amino aci such as aanine that
has ony two issociating groups, there is no ambiguity. The
first pKa (R—COOH) is 2.35 an the secon pKa (R—NH3+) is
9.69. The isoeectric pH (pI) of aanine thus is
OH
For poyprotic acis, one must first ientify the isoionic spe-
cies. For exampe, the pI for aspartic aci is
For ysine, Pi is cacuate from:
O H+ O H+ O
OH O– O–
pK2 = 3.86 pK3 = 9.82
NH3+ (β-COOH) NH3+ (— NH3+) NH2
– – –
O O O
O O
C D
Around pH 3; Around pH 6–8; In strong alkali
net charge = 0 net charge = –1 (above pH 11);
net charge = –2

TABLE 3−4 Typical Range of pK Values for lonizable
Groups in Proteins
Dissociating Group pKa Range
α-Carboxyl 3.5-4.0
Non-α COOH of Asp or Glu 4.0-4.8
Imidazole of His 6.5-7.4
SH of Cys 8.5-9.0
OH of Tyr 9.5-10.5
α-Amino 8.0-9.0
e-Amino of Lys 9.8-10.4
Guanidinium of Arg ~12.0
Simiar consierations appy to a poyprotic acis (eg, pro-
teins), regaress of the number of issociabe groups present.
In the cinica aboratory, knowege of the pI guies seection
of conitions for eectrophoretic separations. Eectrophore-
sis at pH 7.0 wi separate two moecues with pI vaues of 6.0
an 8.0, because the moecue with a pI of 6.0 wi have a net
negative charge, an that with a pI of 8.0 a net positive charge.
Simiar consierations unerie chromatographic separations
on ionic supports such as iethyaminoethy (DEAE) ceuose
(see Chapter 4).
pKa Values Vary With the Environment
The environment of a issociabe group affects its pKa (Table 3–4).
A nonpoar environment, which possesses ess capacity than
water for stabiizing charge species, thus raises the pKa of a
carboxy group making it a weaker aci, but lowers the pKa of
an amino group, making it a stronger aci. Simiary, the pres-
ence of an ajacent oppositely charge group can stabilize, or of
a similarly charge group can destabilize, a eveoping charge.
Therefore, the pKa vaues of the R groups of free amino acis in
aqueous soution (see Tabe 3–1) provie ony an approximate
guie to their pKa vaues when present in proteins. The pKa of
a issociabe R group wi epen on its ocation within a pro-
tein. For exampe, pKa vaues that iverge from aqueous sou-
tion by as much as 3 pH units are common at the active sites of
enzymes, whie a burie aspartic aci of thioreoxin, has a pKa
above 9—a shift of more than 6 pH units!
The Solubility of Amino Acids Reflects
Their Ionic Character
The charges conferre by the issociabe functiona groups of
amino acis ensure that they are reaiy sovate by—an thus
soube in poar sovents such as water an ethano, but inso-
ube in nonpoar sovents such as benzene, hexane, or ether.
Amino acis o not absorb visibe ight an thus are coor-
ess. However, tyrosine, phenyaanine, an tryptophan absorb
high-waveength (250-290 nm) utravioet ight. Because it
CHAPTER 3 Amino Acids & Peptides 21
Optical density of 1.0-mM solutions (1.0-cm path)
6
5
4
Tryptophan
3
2
Tyrosine
1
Phenylalanine
0
240 260 280
Wavelength (nm)
FIGURE 3–6 Ultraviolet absorption spectra of tryptophan,
tyrosine, and phenylalanine.
absorbs utravioet ight about 10 times more efficienty than
either phenyaanine or tyrosine, tryptophan makes the major
contribution to the abiity of most proteins to absorb ight in
the region of 280 nm (Figure 3–6).
THE α-R GROUPS DETERMINE THE
PROPERTIES OF AMINO ACIDS
Each functiona group of an amino aci exhibits a of its
characteristic chemica reactions. For carboxyic aci groups,
these reactions incue the formation of esters, amies, an
aci anhyries; for amino groups, acyation, amiation, an
esterification; an for —OH an —SH groups, oxiation an
esterification. Since gycine, the smaest amino aci, can be
accommoate in paces inaccessibe to other amino acis,
it often occurs where pepties ben sharpy. The hyropho-
bic R groups of aanine, vaine, eucine, an isoeucine an
the aromatic R groups of phenyaanine, tyrosine, an tryp-
tophan occur primariy in the interior of cytosoic proteins.
The charge R groups of basic an aciic amino acis stabi-
ize specific protein conformations via ionic interactions, or
sat briges. These interactions aso function in “charge reay”
systems uring enzymatic cataysis an eectron transport in
respiring mitochonria. Histiine pays unique roes in enzy-
matic cataysis. The pKa of its imiazoe proton permits his-
tiine to function at neutra pH as either a base or an aci
catayst without the nee for any environmentay inuce
shift. The primary thioacoho (—SH) group of cysteine,
whose pKa is 8.3, is an exceent nuceophie an often func-
tions as such uring enzymatic cataysis. For seenocysteine,
its pK3 of 5.2 is 3 pH units ower than that of cysteine. At a
istincty aciic pH, seenocysteine thus shou be the bet-
ter nuceophie. The primary acoho of serine functions as
the active site nuceophie in trypsin an other serine prote-
ases. However, the seconary acoho group of threonine is
not known to serve this roe in cataysis. The —OH groups of
22 SECTION I Structures & Functions of Proteins & Enzymes

serine, tyrosine, an threonine frequenty serve as the points SH

of covaent attachment for phosphory groups that reguate O CH2 H
protein function (see Chapter 9).
C CH N
CH2 N C CH2
Amino Acid Sequence Determines
CH2 H O COO–
Primary Structure
H C NH3+
Amino acis are inke together by peptie bons.
COO–
+
H3N O
H FIGURE 3–7 Glutathione (γ-glutamyl-cysteinyl-glycine). Note
N O–
N the non–α peptide bond that links Glu to Cys.
H
O O
SH

Alanyl Cysteinyl Valine Some Peptides Contain Unusual

The number an order of the amino aci resiues in a
poypeptie constitute its primary structure. Amino acis
present in pepties, cae aminoacyl residues, are referre to
by repacing the ate or ine suffixes of free amino acis with
yl (eg, aanyl, aspartyl, tyrosyl). Pepties are then name as
erivatives of the carboxy termina aminoacy resiue. For
exampe, Lys-Leu-Tyr-Gn is cae ysyl-eucyl-tyrosyl-guta-
mine. The ine ening on the carboxy-termina resiue (eg, gu-
tamine) inicates that its α-carboxy group is not invove in
a peptie bon. Three-etter abbreviations inke by straight
ines represent an unambiguous primary structure. Lines are
omitte when using singe-etter abbreviations.
Gu—Aa—Lys—Gy—Tyr—Aa
E A K G Y A
Prefixes ike tri- or octa- enote pepties with three or eight
residues, respectivey. By convention, pepties are written with
the resiue that bears the free α-amino group at the eft. This
convention was aopte ong before it was iscovere that
pepties are synthesize in vivo starting from the amino-
termina resiue.
Peptide Structures Are Easy to Draw
To raw a peptie, use a zigzag to represent the main chain
or backbone. A the main chain atoms, which occur in
the repeating orer: α-nitrogen, α-carbon, carbony carbon.
Amino Acids
In mammas, peptie hormones typicay contain ony the
20 coon-specifie α-amino acis inke by stanar pep-
tie bons. Other pepties may, however, contain nonprotein
amino acis, erivatives of the protein amino acis, or amino
acis inke by an atypica peptie bon. For exampe, the
amino termina gutamate of gutathione, a tripeptie that
participates in the metaboism of xenobiotics (see Chapter 47)
an the reuction of isufie bons, is inke to cysteine by a
non-α peptie bon that utiizes the sie chain, rather than the
α-, carboxyic aci group (Figure 3–7). The amino termina
gutamate of thyrotropin-reeasing hormone (TRH) is cycize
to pyrogutamic aci, an the carboxy group of the carboxy
termina proy resiue is amiate. The nonprotein amino
acis d-phenyaanine an ornithine are present in the cycic
peptie antibiotics tyrociine an gramiciin S, whie the hep-
tapeptie opiois ermorphin an etophorin in the skin of
South American tree frogs contain d-tyrosine an d-aanine.
The Peptide Bond Has Partial
Double-Bond Character
Athough peptie structures are written as if a singe bon
inke the α-carboxy an α-nitrogen atoms, this bon in fact
exhibits partia oube-bon character:
O O–

Now a a hyrogen atom to each α-carbon an to each C C +

peptie nitrogen, then a an oxygen to the carbony car- N N

bon. Finay, a the appropriate R groups (shae) to each H H

α-carbon atom.
The bon that connects a carbony carbon to the α-nitrogen
N C Cα N C therefore cannot rotate, as this wou require breaking the par-
Cα N C Cα tia oube bon. Consequenty, the O, C, N, an H atoms of
O HC
a peptie bon are coplanar. The impose semirigiity of the
3 H
H peptie bon has important consequences for the manner in
+H N
3 C C N COO– which pepties an proteins fo to generate higher orers
C N C C
H H H CH2 of structure. In Figure 3–8, encircing brown arrows inicate
CH2 O free rotation about the remaining bons of the poypeptie
–OOC OH
backbone.

O R H H
121° 122°
C C N C
120°
0.
117°
110°
14
7 3
N C nm
120° 120°
H O
0.36 nm
FIGURE 3–8 Dimensions of a fully extended polypeptide
chain. The four atoms of the peptide bond are coplanar. Free rota-
tion can occur about the bonds that connect the α-carbon with the
α-nitrogen and with the α-carbonyl carbon (brown arrows). The
extended polypeptide chain is thus a semirigid structure with two-
thirds of the atoms of the backbone held in a fixed planar relationship
to one another. The distance between adjacent α-carbon atoms is
0.36 nm (3.6 Å). The interatomic distances and bond angles, which
are not equivalent, are also shown. (Reproduced with permission
from Pauling L, Corey LP, Branson HR: The structure of proteins: two
hydrogen-bonded helical configurations of the polypeptide chain.
Proc Natl Acad Sci USA. 1951;37(4):205-211.)
Noncovalent Forces Constrain Peptide
Conformations
Foing of a peptie probaby begins coincient with its bio-
synthesis (see Chapter 37). The mature, physioogicay active
conformation refects the coective contributions of the amino
aci sequence, noncovaent interactions (eg, hyrogen bon-
ing, hyrophobic interactions), an the minimization of steric
hinrance between resiues. Common repeating conforma-
tions incue α-heices an β-peate sheets (see Chapter 5).
Peptides Are Polyelectrolytes
The peptie bon is uncharge at any pH of physioogic
interest. Formation of pepties from amino acis is therefore
accompanie by a net oss of one positive an one negative
charge per peptie bon forme at physioogic pH. Pepties
nevertheess are charge owing to their termina carboxy
an amino groups an, where present, their aciic or basic
R groups. As for amino acis, the net charge on a peptie
epens on the pH of its environment an on the pKa vaues
of its issociating groups.
SUMMARY
■ Both d-amino acis an non–α-amino acis occur in nature,
but proteins are synthesize using ony l-α-amino acis.
d-Amino acis o, however, serve metaboic roes, not ony in
bacteria, but aso in humans.
CHAPTER 3 Amino Acids & Peptides 23
■ l-α-Amino acis serve vita metaboic functions in aition
O
to protein synthesis. Exampes incue the biosynthesis of
0.123 nm
urea, heme, nuceic acis, hormones such as epinephrine an
neurotransmitters such as DOPA.
0.
13
2
nm ■ The presence in meteorites of trace quantities of many of the
nm protein amino acis ens creence to the hypothesis that
C 15 N
0. asteroi strikes might have contribute to the eveopment of
0.1 nm
ife on earth.
■ Certain of the l-α-amino acis present in pants an pant
H R H
sees can have eeterious effects on human heath, for
exampe, in athyrism.
■ The R groups of amino acis etermine their particuar
biochemica functions. Amino acis are cassifie as basic,
aciic, aromatic, aiphatic, or sufur-containing base on the
composition an properties of their R groups.
■ The partia oube-bon character of the bon that inks the
carbony carbon an the nitrogen of a peptie rener the four
atoms of the peptie bon coplanar, an hence restrict the
number of possibe peptie conformations.
■ Pepties are often cassifie base on the number of amino aci
resiues present, an are name as erivatives of the carboxy
termina resiue. The primary structure of a peptie is its amino
aci sequence, starting from the amino-termina resiue, a
irection in which pepties actuay are synthesize in vivo.
■ A amino acis possess at east two weaky aciic functiona
groups, R—NH3+ an R—COOH. Many aso possess aitiona
weaky aciic functiona groups such as the phenoic —OH
of tyrosine or the —SH, guaniino, or imiazoe moieties of
cysteine, histiine, an arginine, respectivey.
■ The pKa vaues of a functiona groups of an amino aci or of
a peptie ictate its net charge at a given pH. pI, the isoeectric
pH, is the pH at which a moecue bears no net charge an thus
oes not move in a irect current eectrica fie.
■ The pKa vaues of free amino acis at best ony approximate
their pKa vaues when present in a protein, an can iffer
wiey ue to the infuence of their surrounings in a protein.
REFERENCES
Bener DA: Amino Acid Metabolism, 3r e. Wiey, 2012.
Diaz-Parga P, Goto JJ, Krishnan VV: Chemistry an chemica
equiibrium ynamics of BMAA an its carbamate aucts.
Neurotox Res 2018;33:76.
Koga T, Naraoka H: A new famiy of extraterrestria amino acis
in the Murchison meteorite. Sci Rep 2017;7:636. https://oi.
org/10.1038/s41598-017-00693-9.
Osinski GR, Cocke CS, Pontefract A, et a.: The roe of meteorite
impacts in the origin of ife. Astrobioogy 2020;20:1121.
Secker JM, Lewis SJ: Avances in D-amino acis in neuroogica
research. Int J Mo Sci 2020;21:7325.
Wu G e.: Amino Acids in Nutrition and Health. Springer, 2020.
Yoshimura T, Nishikawa T, Homma H es.: D-Amino Acids:
Physiology, Metabolism, and Application. Springer, 2016.
 C H A P T E R

Proteins: Determination
of Primary Structure
Peter J. Kennelly, PhD, & Victor W. Rodwell, PhD
OBJ E C TI VE S
After studying this chapter,
you should be able to:
4
■ Cite three examples of posttranslational modifications that commonly occur
during the maturation of a newly synthesized polypeptide.
■ Name four chromatographic methods commonly employed for the isolation of
proteins from biologic materials.
■ Describe how electrophoresis in polyacrylamide gels can be used to determine
the purity, subunit composition, relative mass, and isoelectric point of a protein.
■ Describe the basis on which quadrupole and time-of-flight (TOF) spectrometers
determine molecular mass.
■ Describe how the availability of genome sequences has facilitated the
determination of protein primary structure.
■ Explain what is meant by “the proteome” and cite examples of its potential
significance.
■ Describe the advantages and limitations of gene chips as a tool for monitoring
protein expression.
■ Outline three strategies for resolving individual proteins and peptides from complex
biologic samples to facilitate their identification by mass spectrometry (MS).
■ Comment on the contributions of genomics, computer algorithms, and
databases to the identification of the open reading frames (ORFs) that encode
a given protein.

proteolysis (see Chapters 9 and 37), alternates between work-

BIOMEDICAL IMPORTANCE
Proteins are physically and functionally complex macromol-
ecules that perform multiple critically important roles. An
internal protein network, the cytoskeleton (see Chapter 51)
maintains a cell’s shape and physical integrity. Actin and myo-
sin filaments form the contractile machinery of muscle (see
Chapter 51). Hemoglobin transports oxygen (see Chapter 6),
while circulating antibodies defend against foreign invad-
ers (see Chapter 52). Enzymes catalyze reactions that gener-
ate energy, synthesize and degrade biomolecules, replicate
and transcribe genes, process mRNAs, etc (see Chapter 7).
Receptors enable cells to sense and respond to hormones and
other extracellular cues (see Chapters 41 and 42). Proteins
are subject to physical and functional changes that mirror
the life cycle of the organisms in which they reside. A typi-
cal protein is “born” at translation (see Chapter 37), matures
through posttranslational processing events such as selective
ing and resting states through the intervention of regulatory
factors (see Chapter 9), ages through oxidation, deamidation,
etc (see Chapter 57), and “dies” when degraded to its com-
ponent amino acids (see Chapter 28). An important goal of
molecular medicine is to identify biomarkers such as proteins
and/or modifications to proteins whose presence, absence, or
deficiency is associated with specific physiologic states or dis-
eases (Figure 4–1).
PROTEINS & PEPTIDES MUST BE
PURIFIED PRIOR TO ANALYSIS
While new techniques have emerged that permit certain pro-
teins be studied within a living cell, in general the acquisition of
highly purified protein remains essential for the detailed exami-
nation of its physical and functional properties. The isolation of

24
 CHAPTER 4 Proteins: Determination of Primary Structure 25

3' AAAAA 2 Folding 3 Processing

5' SH S
mRNA SH S
Val Val
Gln Gln
Phe Ribosome Phe +−
2H 2e
Asp Asp
Met Met Met-Asp-Phe-Gln-Val
1 Synthesis
4 Covalent modification
(eg, fatty acid acylation)
Trp Phe
Gly His
Glu S
Pro Lys Ala
Asn S
lle Thr Cys
8 “Aging” (eg, Products Substrates
oxidation,
Ub deamidation,
10 Degradation denaturation) 7 Catalysis 5 Translocation
Ub
Ub
Ub 6 Activation
S 9 Ubiquitination S S
S S
S S S

Membrane

FIGURE 4–1 Diagrammatic representation of the life cycle of a hypothetical protein. (1) The life cycle begins with the synthesis on
a ribosome of a polypeptide chain, whose primary structure is dictated by an mRNA. (2) As synthesis proceeds, the polypeptide begins to fold
into its native conformation (blue). (3) Folding may be accompanied by processing events such as proteolytic cleavage of an N-terminal leader
sequence (Met-Asp-Phe-Gln-Val) or the formation of disulfide bonds (S—S). (4) Subsequent covalent modifications may, for example, attach a
fatty acid molecule (yellow) for (5) translocation of the modified protein to a membrane. (6) Binding an allosteric effector (red) may trigger the
adoption of a catalytically active conformation. (7) Over time, proteins get damaged by chemical attack, deamidation, or denaturation, and
(8) may be “labeled” by the covalent attachment of several ubiquitin molecules (Ub). (9) The ubiquitinated protein is subsequently degraded to
its component amino acids, which become available for the synthesis of new proteins.

a specific protein from a natural source in quantities sufficient
for analysis presents a formidable challenge as living organisms
contain thousands of different proteins, each in widely varying
amounts. Thus, obtaining pure protein may require successive
application of multiple separation techniques. Selective pre-
cipitation exploits differences in relative solubility of individual
proteins as a function of pH (isoelectric precipitation), polarity
(precipitation with ethanol or acetone), or salt concentration
(salting out with ammonium sulfate). Chromatographic tech-
niques separate one protein from another based on the differ-
ence in their size and shape (size-exclusion chromatography),
net charge (ion-exchange chromatography), hydrophobicity
(hydrophobic interaction chromatography), or ability to bind
a specific ligand (affinity chromatography).
Column Chromatography
In column chromatography, the stationary phase matrix con-
sists of small beads loaded into a cylindrical container of glass,
plastic, or steel called a column. Liquid-permeable frits con-
fine the beads within the column while allowing the mobile-
phase liquid to flow or percolate through. The stationary
phase matrix can be chemically derivatized to coat each bead’s
surface with the acidic, basic, hydrophobic, or ligand-like
groups required for ion exchange, hydrophobic interaction, or
affinity chromatography. As the mobile-phase liquid emerges
from the column, it is automatically collected as a series of
small portions called fractions. Figure 4–2 depicts the basic
arrangement of a simple bench-top chromatography system.
HPLC—High-Pressure
Liquid Chromatography
First-generation column chromatography matrices consisted
of long, intertwined oligosaccharide polymers shaped into
spherical beads roughly a tenth of a millimeter in diameter.
Unfortunately, their relatively large size perturbed mobile-
phase flow. In theory, resolution could be increased by reduc-
ing particle size. However, the greater pressures required to
overcome the increased resistance of a more tightly packed
matrix crushed the soft and spongy polysaccharide or, later,
polyacrylamide beads. Eventually, small spherical silicon
particles became available that were physically strong and
porous, with a high surface area for attaching various chemical
groups. These particles were then packed into stainless steel
columns capable of withstanding high pressures necessary to
force liquid through the more constricted passages formed by
the smaller, tightly packed beads. The high surface area and
greater uniformity of these silica beads imbue high pressure
liquid chromatography, or HPLC, systems with a resolving
power that is orders of magnitude higher than that of the once
familiar glass columns and their polysaccharide matrices.
26 SECTION I Structures & Functions of Proteins & Enzymes
D E
A A
B B
G
FIGURE 4–2 Components of a typical liquid chromatography
apparatus. Shown are the key components of a programmable liquid
chromatography system consisting of A: reservoirs of mobile-phase
liquids (yellow, light blue), B: microprocessor-controlled pumps
(purple), C: mixing chamber (red), D: injection port for loading analyte
(dark blue); E: glass, metal, or plastic column containing stationary
phase matrix (gray), F: spectrophotometric, fluorometric, refractive
index, or electrochemical detector (orange), and G: fraction collector
for collecting portions, called fractions, of the eluent liquid (green)
in a series of separate test tubes, vials, or wells in a microtiter plate.
The microprocessor can be programmed to pump liquid from only
one reservoir (isocratic elution), to switch reservoirs at some prede-
termined point to generate a step gradient, or to mix liquids from the
two reservoirs in proportions that vary over time to generate either a
multistep or a continuous gradient.
Size-Exclusion Chromatography
Size-exclusion or, as it is sometimes still referred to, gel-
filtration chromatography separates proteins on the basis of their
Stokes radii, which is a function of both molecular mass and
shape. As it rapidly tumbles, an elongated protein effectively
occupies, like a spinning propeller, a larger effective volume
than would a globular protein of the same mass. Size-exclu-
sion chromatography employs porous beads (Figure 4–3)
whose openings are analogous to indentations in a river bank.
If an object floating downstream drifts into an indentation in
the riverbank, its downstream motion is retarded until it drifts
back into the current. Similarly, proteins with Stokes radii
too large to enter the pores (excluded proteins) continuously
remain in the flowing mobile phase, and emerge before pro-
teins able to enter some or all of the pores, where their motion
is retarded. The fraction of the pores accessible to a given pro-
tein, and thus the degree to which their motion is retarded,
increases with decreasing size. Proteins thus emerge from a
gel filtration column in descending order of their Stokes radii.
A B
FIGURE 4–3 Size-exclusion chromatography. A: A mixture of
large molecules (brown) and small molecules (red) is applied to the
top of a gel filtration column. B: Upon entering the column, the small
molecules enter pores in the stationary phase matrix (gray). As the
mobile phase (blue) flows down the column, they lag behind from
the large molecules, which are excluded.
Ion-Exchange Chromatography
In ion-exchange chromatography, proteins interact with the
stationary phase by charge-charge interactions. Proteins with
a net positive charge at a given pH will adhere to beads deriva-
tized with negatively charged functional groups such as car-
boxylates or sulfates (cation exchangers). Similarly, proteins
with a net negative charge will adhere to beads with posi-
tively charged functional groups, typically tertiary or quater-
nary amines (anion exchangers). Nonadherent proteins flow
through the matrix and are washed away. Bound proteins then
can be selectively displaced from the matrix by gradually rais-
ing the ionic strength of the mobile phase, thereby weakening
charge-charge interactions. Purification can be enhanced by
manipulating the pH of the mobile phase, which in turn will
alter the magnitude and even the sign of the net charge of each
component of a protein mixture.
Hydrophobic Interaction
Chromatography
Hydrophobic interaction chromatography separates proteins
based on their tendency to associate with a stationary phase
matrix coated with hydrophobic groups (eg, phenyl Sepharose,
octyl Sephadex). Proteins with exposed hydrophobic surfaces
adhere to the matrix via hydrophobic interactions that are
enhanced by employing a mobile phase of high ionic strength.
After nonadherent proteins are washed away, the polarity of
the mobile phase is decreased by gradually lowering its salt
concentration. If the interaction between protein and station-
ary phase is particularly strong, ethanol or glycerol may be
added to the mobile phase to decrease its polarity and further
weaken hydrophobic interactions.
 CHAPTER 4 Proteins: Determination of Primary Structure 27

Affinity Chromatography S E C H D
Affinity chromatography exploits the high selectivity displayed 111
by enzymes and other proteins for ligands such as substrates,
73
products, cofactors, inhibitors, or analogues thereof. In theory,
when a protein mixture is applied to a matrix derivatized with
a particular ligand, only those proteins that bind that ligand
will adhere. Bound proteins are then eluted either by competi- 48
tion with free, soluble ligand or, less selectively, by disrupt-
ing protein-ligand interactions using high salt concentrations or
other agents. Recombinantly expressed proteins are usually puri-
fied by fusing to their gene an additional segment of DNA that
encodes a convenient ligand-binding domain (see Chapter 7). 34

Protein Purity Is Assessed by 29

Polyacrylamide Gel Electrophoresis
FIGURE 4–5 Use of SDS-PAGE to observe successive purifica-
(PAGE) tion of a recombinant protein. The gel was stained with Coomassie
The most widely used method for determining a sample’s Blue. Shown are protein standards (lane S) of the indicated Mr, in kDa,
crude cell extract (E), cytosol (C), high-speed supernatant liquid (H),
protein composition is SDS-PAGE—polyacrylamide gel elec-
and the DEAE-Sepharose fraction (D). The recombinant protein has a
trophoresis (PAGE) in the presence of the anionic detergent mass of about 45 kDa.
sodium dodecyl sulfate (SDS). Electrophoresis separates
charged biomolecules based on the rates at which they migrate
through a porous matrix in an applied electrical field. For molecules, each bearing a charge of –1, overwhelms the charge
SDS-PAGE, proteins migrate as SDS-polypeptide complexes contributions of the amino acid functional groups endogenous
through a polyacrylamide matrix. Binding of SDS causes most to a typical polypeptide, rendering the charge-to-mass ratio of
polypeptides to unfold or denature. When used in conjunc- each SDS-polypeptide complex approximately equal. Under
tion with 2-mercaptoethanol or dithiothreitol to reduce and these circumstances, the larger the polypeptide, the greater
break disulfide bonds (Figure 4–4), SDS-PAGE separates the the physical resistance its SDS-polypeptide complex encoun-
component polypeptides of multimeric proteins. On average, ters as it moves through the acrylamide matrix. Consequently,
each SDS-polypeptide complex contains one molecule of SDS SDS-PAGE separates most polypeptides based on their rela-
for every two peptide bonds. The large number of anionic SDS tive molecular mass (Mr). Upon completion, the individual
polypeptides trapped in the polyacrylamide gel are visualized
by staining with dyes such as Coomassie Blue (Figure 4–5).
NH
O HN
H
S O Isoelectric Focusing (IEF)
HN S Using polyionic buffers called ampholytes and an applied electric
H
O NH field, a pH gradient can be established within a polyacrylamide
O
matrix. Applied proteins migrate until they reach the region of
the matrix where the pH matches their isoelectric point (pI), the
O SH
pH at which a molecule’s net charge is 0. IEF frequently is used in
HCOOH C2H5 conjunction with SDS-PAGE for two-dimensional electrophore-
OH
sis, which separates polypeptides based on pI in one dimension
and on Mr in the second (Figure 4–6). Two-dimensional electro-
NH phoresis is particularly well suited for separating the components
O within complex mixtures of proteins.
H
HN
SO2–
HN O
O HS H
NH
SANGER WAS THE FIRST TO
O DETERMINE THE SEQUENCE
OF A POLYPEPTIDE
FIGURE 4–4 Oxidative cleavage of adjacent polypeptide While scientists found it relatively simple to determine the
chains linked by disulfide bonds (highlighted in blue) by perfor-
mic acid (left) or reductive cleavage by β-mercaptoethanol (right) amino acid composition of purified proteins, the proportion
forms two peptides that contain cysteic acid residues or cysteinyl of each type of amino acid in the polypeptide, deriving the
residues, respectively. order or sequence of the amino acids proved difficult. The first
28 SECTION I Structures & Functions of Proteins & Enzymes

pH = 3 pH = 10
IEF

N C S

O
+ NH2
H
N
N R
H O
R
SDS
PAGE Phenylisothiocyanate (Edman reagent)
and a peptide

N NH
H
O
H
FIGURE 4–6 Two-dimensional IEF-SDS-PAGE. The gel was N
stained with Coomassie Blue. A crude bacterial extract was first N R
H O
subjected to isoelectric focusing (IEF) in a pH 3–10 gradient. The IEF R
gel was then placed horizontally on the top of an SDS-PAGE gel, and A phenylthiohydantoic acid
the proteins then further resolved by SDS-PAGE. Notice the greatly
improved resolution of distinct polypeptides relative to ordinary SDS-
H+, nitro- H2O
PAGE gel (see Figure 4–5). methane

protein to have its sequence determined was the peptide hor-
mone insulin, an achievement that earned Frederick Sanger a
Nobel Prize in 1958. Mature insulin consists of the 21-residue
A chain and the 30-residue B chain linked by disulfide bonds.
Sanger reduced the disulfide bonds (see Figure 4–4), separated
the A and B chains, and cleaved each chain into smaller and
smaller peptides using the proteolytic enzymes trypsin, chy-
motrypsin, and pepsin followed by incubation with hydro-
chloric acid. Each peptide in the mixture was isolated, treated
with 1-fluoro-2,4-dinitrobenzene (Sanger reagent), and their
amino acid composition determined. Sanger reagent reacts
with the exposed α-amino groups of the amino-terminal resi-
dues, allowing the amino-terminal amino acid of each peptide
to be identified. The ε-amino group of lysine also reacts with
Sanger reagent; but since an amino-terminal lysine reacts with
2 molecules of Sanger reagent, it is readily distinguished from
a lysine from the interior of a peptide. Working from di- and
tripeptides up through progressively larger fragments, Sanger
was able to reconstruct the complete sequence of insulin.
Sanger would later develop the dideoxy method for sequencing
DNA, an accomplishment for which he was awarded a second
Nobel Prize in 1980.
EDMAN DEVISED THE FIRST
PRACTICAL METHOD FOR
PEPTIDE SEQUENCING
Sanger’s labor-intensive approach rendered it prohibitively dif-
ficult to apply to any but the smallest polypeptides. Pehr Edman
discovered a reagent, phenyl isothiocyanate (Edman reagent),
S O
NH2
N NH + N
H R
O R
A phenylthiohydantoin and a peptide
shorter by one residue
FIGURE 4–7 The Edman reaction. Phenyl isothiocyanate
derivatizes the amino-terminal residue of a peptide as a phenyl-
thiohydantoic acid. Treatment with acid in a nonhydroxylic solvent
releases a phenylthiohydantoin, which is subsequently identified by
its chromatographic mobility, and a peptide one residue shorter. The
process is then repeated.
that not only could selectively label the amino-terminal resi-
due of a peptide as its phenylthiohydantoin (PTH) deriva-
tive but, in contrast to Sanger reagent, permitted the PTH
derivative to be removed under mild conditions (Figure 4–7).
The new amino terminal residue could then be treated with
Edman reagent and the process repeated to permit each suc-
cessive residue in a peptide to be derivatized.
While, in theory, one could determine the entire sequence
of a polypeptide using Edman reagent, the heterogeneous
chemical properties of the amino acids meant that every step
in the procedure represented a compromise between effi-
ciency for any particular amino acid or set of amino acids and
the flexibility needed to accommodate all 20. Consequently,
each step in the process operates at less than 100% efficiency,
which leads to the accumulation of polypeptide fragments
with varying N-termini that eventually renders it impossible
 CHAPTER 4 Proteins: Determination of Primary Structure 29

to distinguish the correct PTH amino acid for that position in GENOMICS ENABLES PROTEINS
the peptide from the out-of-phase contaminants. As a result,
the read length for Edman sequencing varies from 5 to 30 TO BE IDENTIFIED FROM SMALL
amino acid residues depending on the quantity and purity AMOUNTS OF SEQUENCE DATA
of the peptide, hardly enough to determine the sequence of a Today the number of organisms for which the complete DNA
typical protein. sequence of their genomes has been determined numbers in
To determine the complete sequence of a polypep- the hundreds of thousands. Thus, for most research scien-
tide several hundred residues in length, a protein must be tists the genetically encoded sequence of the protein(s) with
cleaved into smaller peptides. These were then purified which they are working has already been determined and
and analyzed by Edman sequencing. This yielded multiple can be accessed in a database such as GenBank. To make an
segments of sequence whose location within the protein, unambiguous identification of the amino acid sequence for
with the exception of the amino terminus, was unknown. a protein, sometimes all that is required is the identities of
In order to assemble these short peptide sequences into the as few as five or six consecutive residues. Today mass spec-
complete sequence of the intact polypeptide, it was neces- trometry (MS) has largely replaced the Edman technique as
sary to generate and analyze additional peptides in search the method of choice for protein identification.
of some whose sequences overlapped with one another,
thereby allowing larger segments of sequence to be pieced
together. While the development of automated Edman MASS SPECTROMETRY
sequencers and sophisticated HPLC systems for purifying
peptides often rendered initial acquisition of a fragmen-
CAN DETECT COVALENT
tary, or partial, sequence relatively quick, finding enough MODIFICATIONS
“overlap” peptides to reconstruct the complete sequence of a The development of mass spectrometric (MS) methods that
typical protein via the Edman technique generally required offer superior sensitivity, speed, and capacity to detect post-
large quantities of purified protein and, more importantly, translational modifications (Table 4–1) has led to its replace-
several months or years of additional work. Consequently, ment of the Edman technique as the method of choice for
the determination of a complete amino acid sequence via protein identification.
the Edman method generally was restricted to highly abun-
dant, readily purified proteins.
MASS SPECTROMETERS COME
IN VARIOUS CONFIGURATIONS
MOLECULAR BIOLOGY In a simple, single quadrupole mass spectrometer, a sample
REVOLUTIONIZED is placed under vacuum and allowed to vaporize in the pres-
THE DETERMINATION OF ence of a proton donor to impart a positive charge. An electri-
cal field then propels the cations toward a curved flight tube
PRIMARY STRUCTURE where they encounter a magnetic field, which deflects them at
The sequence for each and every protein in an organism a right angle to their original direction of flight (Figure 4–8).
is encoded in the latter’s genome. By enabling scientists to The current powering the electromagnet is gradually increased
decode the sequences of proteins directly from their structural until the path of each ion is bent sufficiently to strike a detec-
genes, molecular biology revolutionized amino acid sequence tor mounted at the end of the flight tube. For ions of identical
analysis. The reasons for this were twofold. First, recombinant
techniques permit researchers to manufacture a virtually infi-
nite supply of DNA from even minute quantities of template
TABLE 4−1 Mass Increases Resulting From Common
present in the original sample (see Chapter 39), regardless of
Posttranslational Modifications
the encoded protein product’s natural abundance or ease of
isolation. Second, DNA sequencing is far more efficient than Modification Mass Increase (Da)
the Edman technique. Today, automated sequenators routinely Phosphorylation 80
“read” DNA sequences several thousand deoxyribonucleotides
in length. Consequently, if one could determine just a portion Hydroxylation 16
of the sequence of a polypeptide of interest by Edman analysis, Methylation 14
one could synthesize a complementary oligonucleotide probe Acetylation 42
with which to identify the DNA clone containing the gene of
interest. The result was a hybrid approach in which Edman Myristylation 210
chemistry was employed to obtain a partial sequence that was Palmitoylation 238
subsequently exploited to determine the complete amino acid Glycosylation 162
sequence by DNA cloning and sequencing.
30 SECTION I Structures & Functions of Proteins & Enzymes

Accelerator plates
Sample
probe

Flight tube

Sample Chamber

Electromagnet

Variable
power
source

Detector

Vacuum pump

Detector
output

Voltage

FIGURE 4–8 Basic components of a simple mass spectrometer. A mixture of molecules, represented by a red circle, green triangle, and
blue diamond, is vaporized in an ionized state in the sample chamber. These molecules are then accelerated down the flight tube by an electri-
cal potential applied to the accelerator grid (yellow). An adjustable field strength electromagnet applies a magnetic field that deflects the flight
of the individual ions until they strike the detector. The greater the mass of the ion, the higher the magnetic field required to focus it onto the
detector.

net charge, the force required to bend their path to the same
extent is proportionate to their mass.
Time-of-flight (TOF) mass spectrometers employ a
linear flight tube. Following vaporization of the sample in
the presence of a proton donor, an electric field is briefly
applied to accelerate the ions toward a detector at the end
of the flight tube. For molecules of identical charge, the
velocity to which they are accelerated, and hence the time
required to reach the detector, is inversely proportional
to their mass.
Quadrupole mass spectrometers are generally used to
determine the masses of molecules of 4000 Da or less, whereas
TOF mass spectrometers are used to determine the large
masses of complete proteins. Various combinations of mul-
tiple quadrupoles, or reflection of ions back down the linear
flight tube of a TOF mass spectrometer, are used to create
more sophisticated instruments.
Peptides Can Be Volatilized for
Analysis by Electrospray Ionization or
Matrix-Assisted Laser Desorption
For many years, the analysis of peptides and proteins by MS
initially was hindered by difficulties in volatilizing these large
organic molecules. While small organic molecules could be
readily vaporized by heating in a vacuum (Figure 4–9), pro-
teins, oligonucleotides, etc. decomposed on heating. Only
when reliable techniques were devised for dispersing peptides,
proteins, and other large biomolecules into the vapor phase
was it possible to apply MS for their structural analysis and
sequence determination. Three commonly used methods for
dispersion into the vapor phase are electrospray ionization,
matrix-assisted laser desorption and ionization (MALDI),
and fast atom bombardment (FAB). In electrospray ioniza-
tion, the molecules to be analyzed are dissolved in a volatile

Heat Electrospray
ionization
FIGURE 4–9 Three common methods for vaporizing molecules in the sample chamber of a mass spectrometer.
solvent and introduced into the sample chamber in a minute
stream through a charged capillary probe (see Figure 4–9). As the
droplet of liquid emerges into the sample chamber, the charged
probe ionizes the sample while the solvent rapidly disperses,
leaving the macromolecule suspended in the gaseous phase.
Electrospray ionization is frequently used to analyze peptides and
proteins as they elute from an HPLC or other chromatography
column, already dissolved in a volatile solvent. In MALDI, the
sample is mixed with a liquid matrix containing a light-absorbing
dye and a source of protons. In the sample chamber, the mixture
is excited using a laser, causing the surrounding matrix to disperse
into the vapor phase so rapidly as to avoid heating embedded
peptides or proteins (see Figure 4–9). In FAB, large macromol-
ecules dispersed in glycerol or another protonic matrix are bom-
barded by a stream of neutral atoms, for example, xenon, that
have been accelerated to a high velocity. “Soft” ionization by FAB
is frequently applied to volatilize large macromolecules intact.
Peptides inside the mass spectrometer can be broken
down into smaller units by collisions with neutral helium or
argon atoms (collision-induced dissociation) and the masses
of the individual fragments determined. Fortunately, since
peptide bonds are more vulnerable to rupture than carbon-
carbon bonds, the most abundant fragments will differ from
one another by increments of one or two amino acids. Since—
with the exceptions of (1) leucine and isoleucine and (2) glu-
tamine and lysine—the molecular mass of each amino acid is
unique, the sequence of the peptide can be reconstructed from
the masses of its fragments.
Tandem Mass Spectrometry
Complex peptide mixtures can be analyzed, without prior
purification, by tandem MS, which employs the equivalent of
two mass spectrometers linked in series. For this reason, anal-
ysis by tandem instruments is often referred to as MS–MS, or
CHAPTER 4 Proteins: Determination of Primary Structure 31
MALDI
Laser
Feed from chromatography system
MS2. The first mass spectrometer separates individual pep-
tides based on their differences in mass. By adjusting the field
strength of the first magnet, a single peptide can be directed
into the second mass spectrometer, where fragments are gen-
erated and their masses are determined. Alternatively, they
can be held in an electromagnetic ion trap located between
the two quadrupoles and selectively delivered to the second
quadrupole instead of being lost when the first quadrupole is
set to select ions of a different mass.
Tandem MS can be used to screen blood samples from new-
borns for the presence and concentrations of amino acids, fatty
acids, and other metabolites. Abnormalities in metabolite levels
can serve as diagnostic indicators for a variety of genetic dis-
orders, such as phenylketonuria, ethylmalonic encephalopathy,
and glutaric acidemia type 1. In recent years a new generation
of tandem mass spectrometers has appeared, called Q-TOF-MS,
that couple quadrupole (Q) and time-of-flight (TOF) technolo-
gies together in order to harness the best aspects of each.
PROTEOMICS & THE PROTEOME
The Goal of Proteomics Is to Identify
the Entire Complement of Proteins
Elaborated by a Cell Under Diverse
Conditions
While the sequence of the human genome is known, the pic-
ture it provides is both static and incomplete. As genes are
switched on and off and mRNA molecules customized via
alternative splicing (see Chapter 36), the spectrum of proteins
synthesized varies by particular cell type, stages of growth or
differentiation, and in response to external stimuli. Muscle cells
express proteins not expressed by neural cells, and the type of
32 SECTION I Structures & Functions of Proteins & Enzymes
subunits present in the hemoglobin tetramer undergo change
pre- and postpartum. Many proteins undergo posttranslational
modifications during maturation into functionally competent
forms or as a means of regulating their properties. In order to
obtain a more complete and dynamic molecular description of
living organisms, scientists are working to determine the pro-
teome, a term that refers to the identity, abundance, and state
of modification of the entire suite of proteins expressed by an
individual cell at a particular time. Since the proteome for each
component cell of an organism is distinct and changes with
time and circumstances, the ultimate, comprehensive human
proteome constitutes a target of formidable size and complexity.
Simultaneous Determination of
Hundreds of Proteins Is Technically
Challenging
A key goal of proteomics is the identification of proteins whose
levels of expression or modification correlate with medically
significant events. In addition to their potential as diagnostic
indicators, these protein biomarkers may provide important
clues concerning the root causes and mechanisms of a specific
physiologic condition or disease. First-generation proteomics
employed SDS-PAGE or two-dimensional electrophoresis to
resolve the proteins in a biologic sample one from another, fol-
lowed by determination of the amino acid sequence of their
amino terminus by the Edman method. Identities were deter-
mined by searching available polypeptide sequences for pro-
teins that contained a matching N-terminal sequence as well as
a similar Mr and, for 2D gels, pI.
These early efforts were constrained by the limited num-
ber of polypeptide sequences available and the difficulties in
isolating polypeptides from the gels in sufficient quantities
for Edman analysis. Attempts to increase resolving power and
sample yield by increasing the size of the gels were only mar-
ginally successful. Eventually, the development of mass spec-
trometric techniques provided a means for protein sequence
determination whose sensitivity was compatible with electro-
phoretic separation approaches.
Knowledge of the genome sequence of the organism in
question greatly facilitated identification by providing a com-
prehensive set of DNA-encoded polypeptide sequences. It also
provided the nucleotide sequence data from which to con-
struct gene arrays, sometimes called DNA chips, containing
hundreds of distinct oligonucleotide probes. These chips could
then be used to detect the presence of mRNAs containing
complementary nucleotide sequences. While changes in the
expression of the mRNA encoding a protein do not necessarily
reflect comparable changes in the level of the corresponding
protein, gene arrays were both less technically demanding and
more sensitive than first-generation proteomic approaches,
particularly with respect to low abundance proteins.
Second-generation proteomics coupled newly developed
nanoscale chromatographic techniques with MS. The pro-
teins in a biologic sample are first treated with a protease to
hydrolyze them into smaller peptides that are then subject to
reversed-phase, ion-exchange, or size-exclusion chromatogra-
phy to apportion the vast number of peptides into smaller sub-
sets more amenable to analysis. These subsets are analyzed by
injecting the column eluent directly into a double quadrupole
or TOF mass spectrometer. Multidimensional protein iden-
tification technology (MudPIT) employs successive rounds
of chromatography to resolve the peptides produced from the
digestion of a complex biologic sample into several simpler
fractions that can be analyzed separately by MS.
Today, advances in the capability and sensitivity of tandem
mass spectrometers allow them to directly analyze complex
samples. The elimination of prior proteolytic or chromato-
graphic preparation will soon render it feasible to analyze the
proteome of an individual cell.
Bioinformatics Assists Identification
of Protein Functions
The functions of a large proportion of the proteins encoded by
the human genome are presently unknown. Efforts continue to
develop protein arrays or chips for directly testing the poten-
tial functions of proteins on a mass scale. However, while some
protein functions are relatively easy to assay, such as protease
or esterase activity, others are much less tractable. Data mining
via bioinformatics permits researchers to compare amino acid
sequences of unknown proteins with those whose functions
have been determined. This provides a means to uncover clues
to their potential properties, physiologic roles, and mechanisms
of action. Algorithms exploit the tendency of nature to employ
variations of a structural theme to perform similar functions in
several proteins (eg, the Rossmann nucleotide binding fold to
bind NAD(P)H, nuclear targeting sequences, and EF hands to
bind Ca2+). These domains generally are detected in the primary
structure by conservation of particular amino acids at key posi-
tions. Insights into the properties and physiologic role of a newly
discovered protein thus may be inferred by comparing its pri-
mary structure with that of known proteins.
SUMMARY
■ Long amino acid polymers or polypeptides constitute the
basic structural unit of proteins, and the structure of a protein
provides insights into how it fulfills its functions.
■ Proteins undergo posttranslational alterations during their
lifetime that influence their function and determine their fate.
■ By generating a new amino terminus, Edman reagent permitted
the determination of lengthy segments of amino acid sequence.
However, physical and chemical factors generally limited the
number of amino acids that could be reliably identified to 30 or
less, which rendered the complete determination of a protein’s
sequence via the Edman technique a time- and effort- intensive
process.
■ Polyacrylamide gels provide a porous matrix for separating
proteins on the basis of their mobility in an applied direct
current electrical field.

■ The nearly constant ratio at which the anionic detergent SDS
binds proteins enables SDS-PAGE to separate polypeptides
predominantly on the basis of relative size.
■ Because mass is a universal property of all biomolecules and
their derivatives, MS has emerged as a versatile technique
applicable to the determination of primary structure,
identification of posttranslational modifications, and the
detection of metabolic abnormalities.
■ DNA cloning coupled with protein chemistry provided a
hybrid approach that greatly increased the speed and efficiency
for determination of primary structures of proteins.
■ Genomics, the determination of entire polynucleotide
sequences, provides researchers with a blueprint for every
genetically encoded macromolecule in an organism.
■ Proteomic analysis utilizes genomic data to identify the entire
complement of proteins in a biologic sample from partial
amino acid sequence data obtained by coupling protein and
peptide separation methods with sequencing by MS.
■ A major goal of proteomics is the identification of proteins
and their posttranslational modifications whose appearance or
disappearance correlates with physiologic phenomena, aging,
or specific diseases.
■ Bioinformatics refers to the development of computer
algorithms designed to infer the functional properties of
macromolecules through comparison of sequences of novel
proteins with others whose properties are known.
CHAPTER 4 Proteins: Determination of Primary Structure 33
REFERENCES
Aslam B, Basit M, Nisar MA, et al: Proteomics: Technologies and
their applications. J Chromatog Sci 2017;55:182.
Biemann K: Laying the groundwork for proteomics: Mass
spectrometry from 1958 to 1988. J Proteomics 2014;107:62.
Bonner P: Protein Purification, 2nd ed., CRC Press, 2019.
Brown KA, Melby JA, Roberts DS, Ge Y: Top-down proteomics:
Challenges, innovations, and applications in basic and clinical
research. Exp Rev Proteomics 2020;17:719.
Burgess RR, Deutscher MP eds.: Guide to Protein Purification. 2nd
ed., Methods Enzymol, vol. 463, Elsevier, 2009 (Entire volume).
Duarte TT, Spencer CT: Personalized proteomics: The future of
precision medicine. Proteomes 2016;4:29.
Jiang Z, Zhou X, Li R, et al: Whole transcriptome analysis with
sequencing: Methods, challenges and potential solutions.
Cellular Molec Life Sci 2015;72:3425.
Kelly RT: Single-cell proteomics: Progress and prospects. Mol Cell
Proteomics 2020;19:739.
Syu GD, Dunn S, Zhu H: Developments and applications of
functional protein microarrays. Mol Cell Proteomics
2020;6:916.
Van Riper SK, de Jong EP, Carlis JV, et al: Mass spectrometry-based
proteomics: Basic principles and emerging technologies and
directions. Adv Exp Med Biol 2013;990:1.
Wood DW: New trends and affinity tag designs for recombinant
protein purification. Curr Opin Struct Biol 2014;26:54.
 C H A P T E R

Proteins: Higher Orders of

Structure
Peter J. Kennelly, PhD, & Victor W. Rodwell, PhD
OBJ E C TI VE S
After studying this chapter,
you should be able to:
5
■ List the advantages and drawbacks of several common approaches to
classifying proteins.
■ Explain and illustrate the primary, secondary, tertiary, and quaternary structure
of proteins.
■ Identify the major recognized types of protein secondary structures and
explain supersecondary motifs.
■ Describe the kind and relative strengths of the forces that stabilize each order
of protein structure.
■ Describe the information summarized by a Ramachandran plot.
■ Summarize the basic operating principles underlying three key methods
for determining protein structure: X-ray crystallography, nuclear magnetic
resonance spectroscopy, and cryo-electron microscopy.
■ Describe the stepwise process by which proteins fold to attain their native
conformation.
■ Identify the physiologic roles in protein maturation of chaperones, protein
disulfide isomerase, and peptidylproline cis–trans isomerase.
■ Describe the principal biophysical techniques used to study tertiary and
quaternary structure of proteins.
■ Explain how genetic and nutritional disorders of collagen maturation illustrate
the close linkage between protein structure and function.
■ Describe the basic events that underly the molecular pathology of prion diseases.

BIOMEDICAL IMPORTANCE Conversey, many next-generation irect-acting antivira

In nature, form foows function. In orer for a newy synthe-
size poypeptie to mature into a bioogicay functiona pro-
tein capabe of catayzing a metaboic reaction, powering ceuar
motion, or forming the macromoecuar ros an cabes that
provie structura integrity to hair, bones, tenons, an teeth,
it must fo into a specific three-imensiona arrangement, or
conformation. In aition, uring maturation, posttransla-
tional modifications may a new chemica groups or remove
transienty neee peptie segments. Genetic or nutritiona
eficiencies that impee protein maturation are eeterious
to heath. Exampes of the former incue Creutzfet-Jakob
isease, scrapie, Azheimer isease, an bovine spongiform
encephaopathy (“ma cow isease”). Exampes of the atter
incue scurvy (ascorbic aci) an Menkes synrome (Cu).
therapeutics for vira iseases such as hepatitis C or HIV act
by inhibiting the activity of proteases, gycosiases, an pep-
tiy protein cis–trans isomerases that catayze key steps in the
maturation of essentia vira proteins.
CONFORMATION VERSUS
CONFIGURATION
The terms configuration an conformation are often confuse.
Configuration refers to the geometric reationship between a
given set of atoms, for exampe, those that istinguish l- from
d-amino acis. Interconversion of configurational aternatives
requires breaking (an reforming) covaent bons. Conforma-
tion refers to the spatia reationship of every atom in a moecue.

34
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 CHAPTER IV

SPACE AND TIME

§ 1. Are time and space ultimately real or only phenomenal? § 2. The space and
time of perception are limited, sensibly continuous, and consist of a
quantitative element together with a qualitative character dependent on
relation to the here and now of immediate individual feeling. § 3. Conceptual
space and time are created from the perceptual data by a combined process
of synthesis, analysis, and abstraction. § 4. They are unlimited, infinitely
divisible, and there is valid positive ground for regarding them as
mathematically continuous. Thus they form infinite continuous series of
positions. They involve abstraction from all reference to the here and now of
immediate feeling, and are thus homogeneous, i.e. the positions in them are
indistinguishable. They are also commonly taken to be unities. § 5. Perceptual
space and time cannot be ultimately real, because they involve reference to
the here and now of a finite experience; conceptual space and time cannot be
ultimately real, because they contain no principle of internal distinction, and
are thus not individual. § 6. The attempt to take space and time as real leads
to the difficulty about qualities and relations, and so to the indefinite regress. §
7. Space and time contain no principle of unity; there may be many space and
time orders in the Absolute which have no spatial or temporal connection with
each other. § 8. The antinomies of the infinite divisibility and extent of space
and time arise from the indefinite regress involved in the scheme of qualities
and relations, and are insoluble so long as the space and time construction is
taken for Reality. § 9. The space and time order is an imperfect phenomenal
manifestation of the logical relation between the inner purposive lives of finite
individuals. Time is an inevitable aspect of finite experience. How space and
time are transcended in the Absolute experience we cannot say.

§ 1. The problems which arise for the metaphysician from the fact
that the physical order, as it is presented to our senses, consists of
elements having position in space and time, are among the oldest
and most perplexing of all the riddles suggested by the course of our
experience. Adequate discussion of them would demand not only far
more space than we are at liberty to bestow on the topic, but such a
familiarity with the mathematical theory of order and series as is
scarcely possible to any one but an original mathematician. All that
we can do in the present chapter is to deal very superficially with one
or two of the leading problems, more with a view to indicating the
nature of the questions which Metaphysics has to face, than of
providing definite answers to them.
The fundamental problem for Metaphysics is, of course, whether
space and time are ultimate Realities or only appearances; that is,
would the whole system of Reality, as directly apprehended by an
absolute all-containing experience, wear the forms of extension and
succession in time, or is it merely a consequence of the limitations of
our own finite experience that things come to us in this guise? It may
indeed be urged that the contents of the universe must form an order
of some sort for the absolute experience, in virtue of their systematic
unity, but even so it is not clear that order as such is necessarily
spatial or temporal. Indeed, most of the forms of order with which we
are acquainted, both in everyday life and in our mathematical
studies, appear to be, properly speaking, both non-spatial and non-
temporal. Thus, e.g., it is seemingly by a mere metaphor that we
speak of the “successive” integers of the natural number-series, the
“successive” powers of an algebraical symbol, the “successive”
approximations to the value of a continued fraction, in language
borrowed from the temporal flow of events, the true relation involved
being in the first two cases the non-temporal one of logical
derivation, and in the third the equally non-temporal one of
resemblance to an ideal standard. The full solution of the
metaphysical problem of space and time would thus involve (1) the
discrimination of spatial and temporal order from other allied forms of
order, and (2) a decision as to the claim of this special form of order
to be ultimately coherent and intelligible.
The problem thus presented for solution is often, and usually with
special reference to the Kantian treatment of space and time in the
Transcendental Æsthetic, put in the form of the question whether
space and time are subjective or objective. This is, however, at best
a misleading and unfortunate mode of expression which we shall do
well to avoid. The whole distinction between a subjective and an
objective factor in experience loses most of its significance with the
abolition, now effected by Psychology, of the vicious Kantian
distinction between the “given” in perception and the “work of the
mind.” When once we have recognised that the “given” itself is
constituted by the movement of selective attention, it becomes
impossible any longer to distinguish it as an objective factor in
knowledge from the subjective structure subsequently raised upon it.
Kant’s adherence to this false psychological antithesis so completely
distorts his whole treatment of the “forms of intuition,” that it will be
absolutely necessary in a brief discussion like our own to deal with
the subject in entire independence of the doctrines of the Æsthetic,
which unfortunately continue to exercise a disproportionate influence
on the current metaphysical presentment of the problem.[141] It should
scarcely be necessary to point out that the metaphysical questions
have still less to do with the psychological problems, so prominent in
recent science, of the precise way in which we come by our
perception of extension and succession. For Metaphysics the sole
question is one not of the origin but of the logical value of these
ideas.
It is of fundamental importance for the whole metaphysical
treatment of the subject, to begin by distinguishing clearly between
space and time as forms of perception, and space and time as
conceptual forms in which we construct our scientific notion of the
physical order. One chief source of the confusions which beset the
Kantian view is the neglect of Kant and most of his followers to make
this distinction with sufficient clearness. We cannot insist too strongly
upon the point that the space and the time of which we think in our
science as containing the entire physical order, are not space and
time as directly known to us in sense-perception, but are concepts
elaborated out of the space and time of direct perception by a
complicated process of synthesis and analysis, and involving
abstraction from some of the most essential features of the space
and time of actual experience. The following brief discussion may
serve to illustrate the general nature of the relation between the two
forms of space and time, and to exhibit the leading differences
between them.
§ 2. Perceptual Space and Time. Both space and time, as we are
aware of them in immediate perception, are (1) limited. The space
we actually behold as we look out before us with a resting eye is
always terminated by a horizon which has a more or less well-
defined outline; the “specious present,” or portion of duration of
which we can be at any time aware at once as an immediately
presented content, has been shown by elaborate psychological
experimentation to have a fairly well-defined span. Whatever lies
outside this “span of attention” belongs either to the no longer
presented past or to the not yet presented future, and stands to the
sensible present much as the space behind my back to the actually
beheld space before my eyes. Of course, in either case the limits of
the actually presented space or time are not absolutely defined. To
right and left of the line of vision the visible horizon gradually fades
off into the indistinctly presented “margin of consciousness”; the
“sensible present” shades away gradually at either end into the past
and the future. Yet, though thus not absolutely defined, sensible
space and time are never boundless.
(2) Perceptual space and time are both internally sensibly
continuous or unbroken. Concentrate your attention on any lesser
part of the actually seen expanse, and you at once find that it is itself
an expanse with all the characteristics of the wider expanse in which
it forms a part. Space as actually seen is not an aggregate of minima
visibilia or perceptual points in which no lesser parts can be
discriminated; so long as space is visually or tactually perceived at
all, it is perceived as containing lesser parts which, on attending to
them, are found to repeat the characteristics of the larger space. So
any part of the “specious present” to which special attention can be
directed, turns out itself to be a sensible duration. Perceived space is
made of lesser spaces, perceived time of lesser times; the “parts”
not being, of course, actually distinguished from each other in the
original percept, but being capable of being so distinguished in
consequence of varying movements of attention.
(3) On investigating the character of our actual perception of
space and time, it appears to contain two aspects, which we may call
the quantitative and the qualitative. On the one hand, whenever we
perceive space we perceive a certain magnitude of extension,
whenever we perceive time we perceive a longer or shorter lapse of
duration. Different spaces and different times can be quantitatively
compared in respect of the bigness of the extension or the duration
comprised in them. On the other hand, the percept of space or time
is not one of mere extension or duration. It has a very different
qualitative aspect. We perceive along with the magnitude of the
extension the form of its outline. This perception of spatial form
depends in the last resort upon perception of the direction assumed
by the bounding line or lines. Similarly, in dealing with only one
dimension of perceived space, we never perceive length (a spatial
magnitude) apart from the perception of direction (a spatial quality).
The same is true of the perception of time. The lapses of duration we
immediately perceive have all their special direction-quality; the
“specious present” is essentially a simultaneously presented
succession, i.e. a transition from before to after. It must be added
that, in perceptual space and time, the directions thus perceived
have a unique relation to the perceiving subject, and are thus all
qualitatively distinct and irreversible. Direction in space is estimated
as right, left, up, down, etc., by reference to axes through the centre
of the percipient’s body at right angles to each other, and is thus for
any given moment of experience uniquely and unambiguously
determined. Direction in time is similarly estimated with reference to
the actual content of the “focus of consciousness.” What is actually
focal is “now,” what is ceasing to be focal is “past,” what is just
coming to be focal is “future” in its direction.[142]
This is perhaps the most fundamental and important peculiarity of
the space and time of actual perception. All directions in them are
unambiguously determined by reference to the here and now of the
immediate experience of an individual subject. As a consequence,
every individual subject has his own special perceptual space and
time; Geometry and Mechanics depend, to be sure, on the possibility
of the establishment of correspondences between these spatial and
temporal systems, but it is essential to remember that, properly
speaking, the space and time system of each individual’s perception
is composed of directions radiating out from his unique here and
now, and is therefore individual to himself.[143]
§ 3. The Construction of the Conceptual Space and Time Order of
Science. For the purposes of practical life, no less than for the
subsequent object of scientific description of the physical order, it is
indispensably necessary to establish equations or correspondences
between the individual space and time systems of different
percipients. Apart from such correspondences, it would be
impossible for one subject to translate the spatial and temporal
system of any other into terms of his own experience, and thus all
practical intercourse for the purpose of communicating directions for
action would come to an end. For the communication of such
practical directions it is imperative that we should be able mentally to
reconstruct the spatial and temporal aspects of our experience in a
form independent of reference to the special here and now of this or
that individual moment of experience. Thus, like the rest of our
scientific constructions, the establishment of a single conceptual
space and time system for the whole of the physical order is
ultimately a postulate required by our practical needs, and we must
therefore be prepared to face the possibility that, like other
postulates of the same kind, it involves assumptions which are not
logically defensible. The construction is valuable, so far as it does its
work of rendering intercommunication between individuals possible;
that it should correspond to the ultimate structure of Reality any
further than the requirements of practical life demand is superfluous.
The main processes involved in the construction of the conceptual
space and time of descriptive science are three,—synthesis,
analysis, abstraction. (a) Synthesis. Psychologically speaking, it is
ultimately by the active movements of individual percipients that the
synthesis of the individual’s various perceptual spaces into one is
effected. As attention is successively directed, even while the body
as a whole remains stationary, to different parts of the whole
expanse before the eye, the visual space which was originally “focal”
in presentation becomes “marginal,” and the “marginal” focal by a
sensibly gradual transition. When to the movements of head and
eyes which accompany such changes in attention there are added
movements of locomotion of the whole body, this process is carried
further, and we have the gradual disappearance of originally
presented spaces from presentation, accompanied by the gradual
emergence of spaces previously not presented at all. This leads to
the mental construction of a wider space containing all the
individual’s different presentation-spaces, the order in which it
contains them being determined by the felt direction of the
movements required for the transition from one to another.
 As we learn, through intercommunication with our fellows, of the
existence for their perception of perceptual extension never directly
presented to our own senses, the process of synthesis is extended
further, so as to comprise in a single spatial system all the
presentation-spaces of all the individual percipients in an order once
again determined by the direction of the movements of transition
from each to the others. Finally, as there is nothing in the principle of
such a synthesis to impose limits upon its repetition, we think of the
process as capable of indefinite continuance, and thus arrive at the
concept of a space stretching out in all directions without definite
bounds. This unending repetition of the synthesis of perceived
spaces seems to be the foundation of what appears in theory as the
Infinity of Space.
Precisely similar is the synthesis by which we mentally construct a
single time system for the events of the physical order. Now means
for me the content which occupies the centre of attentive interest. As
attention is concentrated on the different stages in the realisation of
an interest, this centre shifts; what was central becomes first
marginal and then evanescent, what was marginal becomes central.
Hence arises the conception of the events of my own inner life as
forming a succession of moments, with a determinate order, each of
which has been a now, or point of departure for directions in
perceptual time, in its turn. As with space so with time, the
intrasubjective intercourse of man with man makes it possible for me
mentally to extend this conceptual synthesis of moments of time so
as to include nows belonging to the experience of others which were
already past before the first now of their experiences which I can
synchronise with a now of my own, and again nows of their
experiences relatively to which the last now which synchronises with
one of my own is past. The indefinite repetition of such a synthesis
leads, as before with space, to the thought of a duration reaching out
endlessly into past and future, and thus gives us the familiar concept
of the Infinity of Time.[144]
(b) Analysis. Equally important is the part played by mental
analysis in the formation of the conceptual space and time system.
As we have already seen, successive attention to lesser parts of a
presented extension, or a presented lapse, reveals within each
lesser part the same structure which belongs to the whole, and thus
establishes the sensible continuity of space and time. In actual fact,
the process of attending successively to smaller and yet smaller
portions of space and time cannot, of course, be carried on
indefinitely, but we can conceptually frame to ourselves the thought
of the indefinite repetition of the process beyond the limits arbitrarily
imposed on it by the span of our own attention. Thus, by an act of
mental analysis, we arrive at the concept of space and time as
indefinitely divisible, or possessed of no ultimately unanalysable last
parts, which is an indispensable pre-requisite of Geometry and
Dynamics.
This indefinite divisibility of conceptual space and time is not of
itself enough, as is often supposed, to establish their continuity in the
strict mathematical sense of the word; their continuity depends upon
the further assumption that whatever divides a series of positions in
space or events in time unambiguously into two mutually exclusive
classes, is itself a position in the space or event in the time series.
This assumption does not seem to be absolutely requisite for all
scientific treatment of the problems of space and time,[145] but is
demanded for the systematic establishment of the correspondence
between the spatial and temporal series and the continuous series of
the real numbers. Moreover, it seems impossible to assign any
positive content to the notion of a something which should bisect the
spatial or temporal order without occupying a position in that order.
Hence we seem inevitably led by the same analytical process which
conducts us to the conception of the spatial and temporal orders as
infinite series to think of them also as continuous series in the strict
sense of the term. The alternative conception of them as
discontinuous, if not absolutely excluded, does not seem to be called
for by any positive motive, and is incompatible with the complete
execution of the purposes which demand application of the number-
series to a spatial or temporal content.
(c) Abstraction. The part played by abstraction in the formation of
the conceptual space and time order out of the data of perception is
often overlooked by theorists, but is of fundamental importance, as
we shall see immediately. We have already learned that the most
significant fact about the time and space order of individual
experience is that its directions are unique, because they radiate out
from the unique here and now of immediate feeling. In the
construction of the conceptual space and time order we make entire
abstraction from this dependence on the immediate feeling of a
subject. Conceptual space contains an infinity of positions, but none
of them is a here; conceptual time an infinity of moments, but none
of them is a now. As the time and space of the conceptual order are
taken in abstraction from the differences between individual points of
view, no one point in either can be regarded as having more claim
than any other to be the natural “origin of co-ordinates” with
reference to which directions are estimated. We shall have repeated
opportunity in the remainder of this chapter to observe how important
are the consequences of this abstraction.
Abstraction also enters in another way into the construction by
which conceptual space and time are created. Actual perceived
space and time are indeed never empty, but always filled with a
content of “secondary” qualities. In other words, they are always one
aspect of a larger whole of fact. Extension is never perceived apart
from some further visual or tactual quality of the extended, temporal
lapse never perceived without some change in presented content,
however slight. But in constructing the conceptual space and time
system, we abstract altogether from this qualitative aspect; we think
solely of the variety of positions and directions in time and space
without taking any account of the further qualitative differences with
which they are accompanied in concrete experience. Thus we come
by the notion of an empty space and an empty time as mere systems
of positions into which various contents may subsequently be put.
Strictly speaking, the notion of an empty space or an empty time is
unmeaning, as the simple experiment of thinking of their existence is
sufficient to show. We cannot in thought successfully separate the
spatial and temporal aspects of experience from the rest of the
whole to which they belong and take them as subsisting by
themselves, any more than we can take timbre as subsisting apart
from musical pitch or colour-tone from saturation. We can, however,
confine our attention to the spatial-temporal system of positions
without taking into account the special secondary properties of the
extended and successive. It is from this logical abstraction that the
illusion arises when we imagine an empty set of spatial and temporal
positions as having first to exist in order that they may be
subsequently “filled” with a variety of contents.[146]
§ 4. Characteristics of the Conceptual Time and Space Order. The
following characteristics of the conceptual space and time created by
the construction we have just examined, call for special notice.
Conceptual space and time are necessarily taken, for reasons
already explained, to be unlimited, and indefinitely divisible. Though
it does not seem inevitable that they should be continuous, we
appear to be unable to attach any positive meaning to the notion of
their discontinuity, and, in the practical need for the application to
them of the complete number-series, we have a valid positive ground
for taking them as continuous. But space and time are thus resolved,
in the process of their conceptual construction, into continuous
infinite series of which the terms are spatial and temporal positions
or points. Unlike the parts of perceptual space and time, these
conceptual terms are not themselves spaces or times, as they
contain no internal multiplicity of structure. Conceptual space and
time are thus not wholes or aggregates of parts, but systems of
relations between terms which possess no quantitative character.
Between any two terms of the spatial, or again of the temporal,
series there is one unique relation, which is completely determined
by the assignment of the terms, their distance. In the temporal
series, which has only one dimension, you can only pass from any
one given term to any other through a series of intermediate terms
which is once and for all determined when the initial and final terms
are given, hence nothing is required beyond the terms themselves to
fix their distance. The spatial series is multi-dimensional, i.e. you can
pass from any one term in it to any second by an indefinite variety of
routes through intermediate terms, but it is still true that there is one
and only one such route which is completely determined when the
terms in question are known, namely, the straight line passing
through both. This straight line constitutes the unique distance of the
two points from each other.[147] Thus the genuine concept of which
those of space and time are species is not that of magnitude or
quantity, but of serial order.
 Further, and this is a point of fundamental difference between
conceptual space and time, and the spaces and times of immediate
perception, any one position in either order, taken by itself, is
qualitatively indistinguishable from any other. All points of space, all
moments of time, are alike, or, as it is also phrased, conceptual
space and time are homogeneous throughout. It is not until you take
at least two terms of the spatial or temporal series and consider the
relation they determine, that distinction becomes possible. This
homogeneity of conceptual space and time is an inevitable
consequence of the abstraction from the immediate feelings of the
individual subject of experience involved, as we saw, in the process
of their construction. In our actual perception of spatial and temporal
extension, that part of perceived space and time which stands in
direct unity with immediate feeling is qualitatively distinguished as
the here and now from all the rest, and thus does not depend upon
the specification of a second spatial or temporal position for its
recognisability. Here is where I am, now is this felt present. And
similarly, every other part of the actually presented space and time
gets a unique qualitative character from its special relation to this
here and now; it is right or left, behind or in front, before or after.
When we abstract altogether from the unique relation with individual
experience which thus makes the here and now of perception, as we
do in constructing our conceptual space and time order, every
position alike becomes the mere possibility of a here or a now, and
as such mere possibilities the various positions are indistinguishable.
Practically, this homogeneity is important as the indispensable
condition for the quantitative comparison of different portions of
extension or duration.
An apparently inevitable consequence of the homogeneity of
conceptual space and time is the relativity of spatial and temporal
position. As we have seen, positions in conceptual space and time
are not distinguishable until you take them in pairs. In other words, to
fix one position in space or one date you have to give its relation to
another position or date, and similarly to fix this you must specify a
third, and so on indefinitely. To say where A is means to say how you
get to it from B, and B again is only known by the way it is reached
from C, and so on without end. Logically, this is a simple
consequence of the nature of space and time as conceptually
analysed into endless series. To specify any term in the series you
must give the unique relation it bears to some other term, its logical
distance. And, in a series which has neither first nor last term, this
second term cannot be defined except by its logical distance from a
third. In actual perception this difficulty is avoided, owing to the fact
that immediate feeling gives us the here and now from which all our
directions are measured. But in conceptual space or time there is
nothing to distinguish any one here which we may take as our “origin
of co-ordinates,” or any one now which we take as our present from
any other, and hence the endless regress seems inevitable.
It follows, of course, that in conceptual space and time there is no
principle by which to distinguish different directions. In perception
they can be distinguished as right and left, up and down, and so
forth. But since what is right to one percipient is left to another, in
conceptual space, where complete abstraction is made from the
presence of an individual percipient, there is neither right nor left, up
nor down, nor any other qualitative difference between one direction
and another, all such differences being relative to the individual
percipient. When we wish to introduce into conceptual space
distinctions between directions, we always have to begin by
arbitrarily assigning some standard direction as our point of
departure. Thus we take, e.g., an arbitrarily selected line ——— as
A B
such a standard for a given plane, and proceed to distinguish all
other directions by the angle they make with A B and the sense in
which they are estimated (whether as from B to A or from A to B).
But both the line A B and the difference of sense between A B and B
A can only be defined by similar reference to some other standard
direction, and so on through the endless regress.
Similarly with conceptual time. Here, as there is only one
dimension, the difficulty is less obvious, but it is no less real. In
conceptual time there is absolutely no means of distinguishing
before from after, past from future. For the past means the direction
of our memories, the direction qualified by the feeling of “no longer”;
the future is the direction of anticipation and purposive adaptation,
the direction of “not yet.” And, apart from the reference given by
immediate feeling to the purposive life of an individual subject, these
directions cannot be discriminated. In short, conceptual time and
space are essentially relative, because they are systems of relations
which have no meaning apart from qualitative differences in the
terms which they relate; while yet again, for the purpose of the
conceptual construction which yields them, the terms have to be
taken as having no character but that which they possess in right of
the relations.[148]
One other feature of the space and time construction is sufficiently
important to call for special mention. Space and time are commonly
thought of as unities of some kind. All spatial positions, it is usually
assumed, fall within one system of space-relations; all dates have
their place in one all-inclusive time. This character of unity completes
the current conception of the spatial and temporal order. Each of
those orders is a unity, including all possible spatial or temporal
positions; each is an endless, infinite, continuous series of positions,
which all are purely relative. There are other peculiarities, especially
of the current concepts of space, with which it is not necessary to
deal here, as they are of an accidental kind, not arising out of the
essential nature of the process by which the conception is
constructed. Thus it is probably a current assumption that the
number of dimensions in space is three and no more, and again that
the Euclidean postulate about parallels is verified by its constitution.
As far as perceptual space is concerned, those assumptions
depend, I presume, upon empirical verification; there seems to be no
reason why they should be made for the conceptual space-order,
since it is quite certain that a coherent science of spatial relations
can be constructed without recourse to them.[149]
§ 5. The question now is, whether the whole of this spatial and
temporal construction is more than imperfect, and therefore
contradictory, appearance. I will first state in a general form the
arguments for regarding it as appearance, and then proceed to
reinforce this conclusion by dealing with some special difficulties.
Finally, I propose to ask whether we can form some positive
conception of the higher order of Reality of which the spatial and
temporal series are phenomenal.
 That the space and time order is phenomenal and not ultimate,
can, I think, be conclusively shown by a general argument which I
will first enunciate in principle and then develop somewhat more in
detail. An all-comprehensive experience cannot apprehend the detail
of existence under the forms of space and time for the following
reason. Such an experience could be neither of space and time as
we perceive them, nor of space and time as we conceptually
reconstruct them. It would not be of perceptual space and time,
because the whole character of our perceptual space and time
depends upon the very imperfections and limitations which make our
experience fragmentary and imperfect. Perceptual space and time
are for me what they are, because I see them, so to say, in
perspective from the special standpoint of my own particular here
and now. If that standpoint were altered, so that what are actually for
me there and then became my here and now, my whole outlook on
the space and time order would suffer change. But the Absolute
cannot look at the space and time order from the standpoint of my
here and now. For it is the finitude of my interests and purposes
which confine me in my outlook to this here and now. If my interests
were not bound up in the special way in which they are with just this
special part or aspect of the life of a wider whole, if they were co-
extensive with the life of that whole, every place and every time
would be my here and now. As it is, here is where my body is, now is
this particular stage in the development of European social life,
because these are the things in which I am primarily interested. And
so with all the other finite experiences in which the detail of the
absolute experience finds expression. Hence the absolute
experience, being free from the limitations of interest which condition
the finite experiences, cannot see the order of existence from the
special standpoint of any of them, and therefore cannot apprehend it
under the guise of the perceptual space and time system.
Again, it cannot apprehend existence under the forms of space
and time as we conceptually reconstruct them. For Reality, for the
absolute experience, must be a complete individual whole, with the
ground of all its differentiations within itself. But conceptual space
and time are constructed by deliberate abstraction from the relation
to immediate experience implied in all individuality, and
consequently, as we have just seen, they contain no real principle of
internal distinction, their constituent terms being all exactly alike and
indistinguishable. In short, if the perceptual time and space systems
of our concrete experience represent individual but imperfect and
finite points of view, the conceptual space and time of our scientific
construction represents the mere abstract possibility of a finite point
of view; neither gives a point of view both individual and infinite, and
neither, therefore, can be the point of view of an absolute
experience. An absolute experience must be out of time and out of
space, in the sense that its contents are not apprehended in the form
of the spatial and temporal series, but in some other way. Space and
time, then, must be the phenomenal appearance of a higher reality
which is spaceless and timeless.
§ 6. In principle, the foregoing argument appears to me to be
complete, but, for the sake of readers who care to have its leading
thought more fully developed, it may be re-stated thus. Perceptual
space and time cannot be ultimately real as they stand. They are
condemned already by the old difficulty which we found in the notion
of reality as made up of qualities in relation. Perceptual space and
time are aggregates of lesser parts, which are themselves spaces
and times; thus they are relations between terms, each of which
contains the same relation once more in itself, and so imply the now
familiar indefinite regress.[150] Again, when we try in our conceptual
space and time construction to remedy this defect by reducing space
and time altogether to mere systems of relations, the difficulty turns
out to have been merely evaded by such a process of abstraction.
For, so long as we keep rigidly to our conceptual construction, the
terms of our relations are indistinguishable. In purely conceptual
space and time, as we have seen, there is no possibility of
distinguishing any one direction from any other, since all are
qualitatively identical.
Indeed, it is obvious from first principles that when the sets of
terms between which a number of relations of the same type holds
are indistinguishable, the relations cannot be discriminated. To
distinguish directions at all, we must, in the end, take at least our
starting-point and one or more standard directions reckoned from it
—according to the number of dimensions with which we are dealing
—as independently given, that is, as having recognisable qualitative
differences from other possible starting-points and standard
directions. (Thus, to distinguish before and after in conceptual time,
you must at least assume some moment of time, qualitatively
recognisable from others, as the epoch from which you reckon, and
must also have some recognisable qualitative distinction between
the direction “past” and the direction “future.”) And with this reference
to qualitative differences we are at once thrown back, as in the case
of perceptual time and space, on the insoluble old problem of Quality
and Relation. The assumed starting-point and standard directions
must have qualitative individuality, or they could not be
independently recognised and made the basis for discrimination
between the remaining directions and positions: yet, because of the
necessary homogeneity of the space and time of conceptual
construction, they cannot have any such qualitative individuality, but
must be arbitrarily assumed. They will therefore themselves be
capable of determination only by reference to some other equally
arbitrary standard, and thus we are once more committed to the
indefinite regress. The practical usefulness of these constructions
thus depends on the very fact that we are not consistent in our use
of them. In all practical applications we use them to map out the
spatial and temporal order of events as seen in perspective from a
standpoint which is, as regards the conceptual time and space order
itself, arbitrary and indistinguishable from others.
§ 7. Instead of further elaborating this general argument, a task
which would be superfluous if its principle is grasped, and
unconvincing if it is missed, I will proceed to point out one or two
special ways in which the essential arbitrariness of the spatial and
temporal construction is strikingly exemplified. To begin with, a word
may be said about the alleged unity of space and time. It is
constantly taken for granted, by philosophers as well as by practical
men, that there can be only one spatial and one temporal order, so
that all spatial relations, and again all temporal relations, belong to
the same system. Thus, if A has a spatial relation to B and C to D, it
is assumed that there must be spatial relations between A and C, A
and D, and B and C, B and D. Similarly if A is temporally related with
B, and C with D. This view is manifestly presupposed in the current
conception of Nature, the “physical universe,” the “physical order,” as
the aggregate of all processes in space and time. But there seems to
be no real logical warrant for it. In principle the alleged unity of all
spatial and temporal relations might be dismissed, on the strength of
the one consideration that space and time are not individual wholes,
and therefore can contain no principle of internal structural unity.
This is manifest from the method by which the space and time of our
conceptual scheme have been constructed. They arose, as we saw,
from the indefinite repetition of a single type of relation between
terms in which we were unable to find any ultimately intelligible
principle of internal structure. But unity of structure cannot be
brought into that which does not already possess it by such mere
endless repetition. The result of such a process will be as internally
incoherent and devoid of structure as the original data. Hence space
and time, being mere repetitions of the scheme of qualities in
relation, cannot be true unities.
This becomes clearer if we reflect on the grounds which actually
warrant us in assigning position in the same space and the same
time to a number of events. For me A and B are ultimately in the
same space when there is a way of travelling from A to B; they are in
the same time when they belong to different stages in the
accomplishment of the same systematic purposes. Thus in both
cases it is ultimately from relation to an identical system of purposes
and interests that different sets of positions or events belong to one
space or one time. The unity of such a space or time is a pale
reflection in abstract form of the unity of a life of systematic purpose,
which is one because it has unique individual structure. It is in this
way, from the individual unity of the purpose and interests of my
ordinary waking life, that I derive the right to refer its experiences to
a single space and time system. Similarly, it is in virtue of the
inclusion of my own and my fellow-men’s purposes in a wider whole
of social systematic purpose that I can bring the space and time
relations of their experience into one system with my own. And
again, the sensible occurrences of the physical order belong to one
space and time with the space and time relations of human
experience, because of the varying ways in which they condition the
development of our own inner purposive life. But there are cases,
even within our own conscious life, where this condition appears to
be absent, and in these cases we do not seem to be able to make
intelligible use of the conception of a single time or a single space.
Take the case of our dreams. The events of my dreams stand in
spatial and temporal relations within the dream itself, but there would
be no sense in asking what are the spatial relations between the
places seen in my dreams and the places marked on the map of
England; or what are again the temporal relations between the
events of last night’s dream and those of this morning, or those of
the dreams of last week. Precisely because there is usually no
systematic identity of purpose connecting the dream with the waking
life or with other dreams, the time and space of the dream have no
position with respect to the time and space system of waking life, nor
those of one dream with relation to those of another.[151] Of course, it
may be said that the dream-space and dream-time are “imaginary,”
but the problem cannot be got rid of by the use of an epithet. To call
them imaginary is merely to say that they are not systematically
connected with the time and space of waking life, not to disprove
their genuineness as actual space and time constructions.
Similarly, if there are intelligent purposes of which our human
purposive life is debarred from taking account as such, as we urged
that there must be behind the phenomenal physical order, the time
and space within which those purposes are conceived and executed
would have no place in our spatial and temporal system. The
phenomenal events of the physical order would fall within our
system, but not the life of inner purpose of which that order is the
manifestation to our senses. Ultimately, in fact, all spaces and all
times could only form one spatial and temporal system on condition
that the infinite absolute experience views all its contents in spatial
and temporal form; then the various space and time systems
corresponding to the purposes of the various groups of finite
individuals would finally, for the infinite individual, form one great
system of time and space relations. But we have already seen that
the infinite experience cannot comprehend its contents in spatial or
temporal forms.
We infer, then, that there may be—indeed, if our interpretation of
the physical order is valid, there must be—a plurality of spaces and
times within the Real. Within any one such space or time all its
members are spatially and temporally interrelated, but the various
spaces are not themselves related in space, nor the various times
before or after one another in time. Their relation is the purely logical
one of being varying modes of the expression in a finite detail of the
underlying nature of the ultimate Reality.[152] For the absolute
experience they must be all at once and together, not in the sense of
being in “one space and time,” but in the sense of forming together
the systematic embodiment of one coherent ground or principle.
§ 8. Similar consequences, as to the phenomenal character of
space and time, follow from the consideration of the familiar Kantian
antinomies founded upon the concept of spatial and temporal infinity.
Space and time must be externally boundless and internally
indefinitely divisible, and yet again cannot be either. Freed from
unessential accessories, the argument for either side of the antinomy
may be stated thus. Space and time must be boundless because all
spatial and temporal existence means spatial and temporal relation
to a second term, itself similarly related to a third term. For precisely
the same reason both must be indefinitely divisible. Yet again, they
can be neither, since only the individual exists, and within such an
interminable network of relations between terms which are nothing
but the supporters of these relations there is no principle of individual
structure.[153] Thus the Kantian antinomies are a simple consequence
of the old difficulty about quality and relation. Space and time must
be mere relations, and the terms of those relations therefore
qualitatively indistinguishable; again, since they are relations they
cannot be relations between nothings or, what is the same thing,
between terms with no individual character. As in all cases where the
problem of relation and quality arises, it then conducts us to the
indefinite regress.
So long as we continue to look upon space and time as real, we
have therefore to choose between two equally illogical alternatives.
We must either arbitrarily refuse to continue the indefinite regress
beyond the point at which its difficulties become apparent, as is done
by the assertion that space and time have finite bounds or indivisible
parts, or we must hold that the absolute experience actually
achieves the summation of an unending series. With the recognition
that space and time are phenomenal, the result of a process of
construction forced on us by our practical needs, but not adequately
corresponding to the real nature of individual existence, the difficulty
disappears. Both sides of the antinomy become relatively true, in the
sense that for our practical purposes we must be content to adopt
now the one and again the other; both become ultimately untrue in
the sense that space and time, being constructions of our own, are
really neither finite nor infinite series, but are the one or the other
according to the purposes for which we use our construction.
§ 9. If spatial and temporal position and direction must thus in the
end be appearance, phenomenal of some more individual reality, we
have finally to ask, Of what are they the appearance? It is not
enough to say “of ultimate Reality,” or “of the Absolute.” Ultimately
this is, no doubt, true of space and time, as it is of everything else,
but we desire further to know if they are not proximately the
appearance of some special features of the inner physical life of the
lesser individuals which compose the Absolute. We naturally look for
some third term, in the nature of finite individuality, to mediate
between the structureless abstract generality of space and time
relation, and the perfect individual structure of the spaceless and
timeless Absolute Individual. We want, in fact, to connect the spatial
and temporal form which our experience wears, with some
fundamental aspect of our nature, as beings at once individual and
finite.
Nor is it particularly difficult to make the connection. When we
remember that space and time, as they actually condition our
perception and movement, are the space and time which radiate out
from an unique here and now of immediate feeling, it is fairly evident
that the spatial and temporal aspect of our experience is, as already
suggested, a consequence of that limitation of our attentive interests
which constitutes our finitude. It is the narrowness of my interests, or
at least of those which are sufficiently explicit to rise into the “focus”
of consciousness, that is reflected in the distinction of my here from
all the theres which are around me. Here is where my body is,
because of the specially intimate connection of the realisation of my
interests and purposes with those events in the phenomenal physical
order which I call the state of my body. Were my interests widened