Biochemistry
College of Allied Health Sciences
Bachelor of Science in Medical Laboratory Science
First Semester, A.Y. 2022-2023
[TRANS] PROTEINS
PROTEINS:
 Proteins are diverse and abundant biomolecules, making
up more than 50% of a cell's dry weight. They play a central
role in various aspects of cell structure and function, and
their diversity is encoded in the genetic information of cells,
specifically in the DNA sequence.
THREE BASIC CLASSES OF PROTEINS:
 Proteins can be categorized into three primary classes
based on shape and solubility: fibrous, globular, and
membrane proteins. Each class has distinct structural and
solubility characteristics.
o Fibrous Proteins: Fibrous proteins are characterized
by their relatively simple, elongated, and linear
structures. They often serve structural roles in cells,
providing support and stability. Some examples of
fibrous proteins include collagen, which is a major
component of connective tissues like skin and
tendons, and keratin, found in hair and nails. Fibrous
proteins are typically insoluble in water and dilute
salt solutions due to their regular and extended
structure.
o Globular Proteins: Globular proteins have a roughly
spherical shape. They are compactly folded, with
hydrophobic (water-repelling) amino acid side chains
located in the interior of the molecule and hydrophilic
(water-attracting) side chains exposed on the
outside, interacting with the surrounding solvent,
usually water. This globular shape makes globular
proteins highly soluble in aqueous solutions. Most of
the soluble proteins found in the cytoplasm of cells,
including enzymes, are globular in shape.
o Membrane Proteins: Membrane proteins are
associated with various cellular membrane systems.
To interact with the nonpolar environment within cell
membranes, membrane proteins have hydrophobic
amino acid side chains oriented outward. This
orientation allows them to integrate into the lipid
bilayers of membranes. As a result, membrane
proteins are generally insoluble in aqueous solutions
but can be solubilized in solutions containing
detergents. Membrane proteins typically have fewer
hydrophilic amino acids exposed compared to
cytosolic proteins.
STRUCTURE OF PROTEINS
 Primary Structure:
o The primary structure of a protein refers to its specific
amino acid sequence. This sequence is essential
because it determines all higher levels of protein
structure.
 Secondary Structure:
o Secondary structure involves the folding of the
polypeptide chain into characteristic helical or
pleated segments due to hydrogen-bonding
interactions between adjacent amino acid residues.
 Tertiary Structure:
o Tertiary structure is achieved when polypeptide
chains bend and fold to assume a compact three-
dimensional shape, giving the protein its unique
three-dimensional conformation.
 Quaternary Structure:
o Quaternary structure is relevant for proteins
consisting of two or more interacting polypeptide
chains. Each chain is referred to as a subunit, and
the quaternary structure describes how these
subunits interact with each other.
 Noncovalent Forces:
o Noncovalent forces, such as hydrogen bonds, ionic
interactions, van der Waals forces, and hydrophobic
interactions, drive the formation of secondary and
higher orders of protein structure. These forces are
key for maintaining the protein's overall
conformation.
 Conformation:
o Conformation refers to the overall three-dimensional
structure of a protein. It should not be confused with
configuration, which relates to the geometric
possibilities for a set of atoms.
PROTEIN SEPARATION METHODS:
 Protein chemists use various methods to separate and
purify proteins. These methods can be based on
differences in size, electrical charge, or specific recognition
properties of a protein.
 Size Separation:
o Techniques like size exclusion chromatography,
ultrafiltration, and ultracentrifugation separate
proteins based on their size.
 Charge Separation:
o Proteins' ionic properties are determined by their
amino acid side chains and can be used for
separation methods like ion exchange
chromatography and electrophoresis.
 Isoelectric Point:
o The isoelectric point is the pH at which a protein's
positive and negative electrical charges balance,
leading to minimal electrostatic repulsion and
increased likelihood of coalescence and
precipitation.
 Solubility Properties:
o Protein solubility is influenced by factors such as
ionic strength. Salting-in and salting-out phenomena
describe how the presence and concentration of salt
ions affect protein solubility.
 Hydrophobic Interaction Chromatography:
o This technique exploits the hydrophobicity of
exposed nonpolar amino acid side chains on a
protein's surface to aid in purification.
ESTIMATION OF PROTEIN CONCENTRATIONS:
 Biochemists often need to determine protein concentrations
in biological samples, which can be complex and contain
proteins of varying molecular weights.
 Traditional protein estimations cannot be expressed on a
molar basis due to the diverse nature of proteins.
 The chemical properties of proteins differ based on their
amino acid composition, nitrogen or sulfur content, and the
presence of various functional groups.
METHODS FOR ESTIMATING PROTEIN
CONCENTRATIONS:
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 Several methods rely on the reduction of Cu²⁺ ions to Cu⁺
by proteins' oxidizable components, such as cysteine or
phenols and indoles of tyrosine and tryptophan.
o Examples of assays include the bicinchoninic acid
(BCA) method and dye binding assays like the
Bradford assay, which use color changes to estimate
protein concentration.
o The Bradford assay employs Coomassie Brilliant
Blue G-250 dye, which binds noncovalently to
proteins.
 Typical Protein Purification Scheme:
o Protein purification aims to isolate a specific protein
to a homogeneous state with acceptable yield.
o A protein's specific activity can increase significantly
during purification, indicating enrichment.
 Acid Hydrolysis:
o Peptide bonds of proteins can be hydrolyzed by
strong acid to release individual amino acids.
o Acid hydrolysis is preferred for analyzing the amino
acid composition of proteins, as it does not lead to
racemization or severe destruction of certain amino
acids.
o Hydrolysis is typically performed with 6 N HCl at
elevated temperature, although it has limitations
related to specific amino acids.
 Chromatographic Separation of Amino Acids:
o The mixture of amino acids generated by hydrolysis
can be separated into individual amino acids using
chromatography methods, such as ion exchange
chromatography or high-pressure liquid
chromatography (HPLC).
o Automated instruments known as amino acid
analyzers are used for this purpose.
AMINO ACID COMPOSITIONS OF PROTEINS:
 Amino acids within proteins rarely occur in equimolar ratios,
indicating that proteins are not composed of repeating
amino acid arrays.
 The amino acid composition can vary significantly between
different proteins.
 Amino acid analysis does not provide information about the
sequence of amino acids in a polypeptide chain.
DETERMINING THE PRIMARY STRUCTURE OF A
PROTEIN
 The primary structure of a protein is defined by the specific
sequence of amino acid residues in its polypeptide chain(s).
 The amino acid sequence is read from the N-terminal
(amino-terminal) end to the C-terminal (carboxy-terminal)
end.
 Early protein sequencing was pioneered by Frederick
Sanger, who determined the amino acid sequences of
insulin's polypeptide chains. This demonstrated that
proteins have a fixed composition and a defined sequence.
 Today, the amino acid sequences of numerous proteins are
determined through various methods, including mass
spectrometry and gene-based deduced sequences.
STEP 1: SEPARATION OF POLYPEPTIDE CHAINS
 If the protein is a heteromultimer with multiple types of
polypeptide chains, it needs to be dissociated into its
individual chains.
 Techniques like exposure to extreme pH, urea, guanidinium
hydrochloride, or high salt concentrations are used for
dissociation.
 Individual polypeptide chains are isolated based on size
and charge differences.
 Crosslinks between chains need to be cleaved if present
STEP 2: CLEAVAGE OF DISULFIDE BRIDGES
 Various methods for cleaving disulfide bridges are
discussed, with a focus on avoiding the reformation of
disulfide bonds.
 Oxidation with performic acid or reduction with sulfhydryl
compounds followed by alkylation are used.
STEP 3: A. N-TERMINAL ANALYSIS
 Edman degradation is the preferred method for identifying
the amino acid at the N-terminal end of a protein.
 This method allows sequential identification of residues
starting from the N-terminus.
 It involves the reaction of the N-terminus with
phenylisothiocyanate, excision of the N-terminal residue,
and identification of the PTH derivative.
 Automated instruments, Edman sequenators, can carry out
repeated rounds of Edman degradation.
STEP 3: B. C-TERMINAL ANALYSIS
 For identifying the C-terminal residue, enzymatic methods
are commonly used.
 Different carboxypeptidases like A, B, C, and Y are used
depending on the specific C-terminal residues.
 The enzymatic approach is adapted to an automated
protocol similar to Edman sequencing.
STEPS 4 AND 5: FRAGMENTATION OF THE
POLYPEPTIDE CHAIN
 The aim is to produce fragments suitable for sequence
analysis.
 Trypsin, chymotrypsin, and other endopeptidases are used
for specific proteolysis.
 Cyanogen bromide and other chemical methods for
fragmentation are also discussed.
 The choice of enzyme and method depends on the specific
goals of the analysis.
STEP 6: RECONSTRUCTION OF THE OVERALL
AMINO ACID SEQUENCE
 Fragments from different cleavage procedures are
compared to establish continuity of the overall amino acid
sequence.
 This step involves aligning and combining the sequence
data from different fragments to reconstruct the complete
protein sequence.
 Amino Acid Sequence of a Protein Can Be Determined by
Mass Spectrometry
MASS SPECTROMETRY
 Mass spectrometry is used to determine the mass-to-
charge (m/z) ratio of ionized protein molecules.
 Electrospray mass spectrometry and tandem mass
spectrometry (MS/MS) are explained.
 Tandem MS allows sequencing of proteins by analyzing
fragments.
 Peptide mass fingerprinting is used to identify proteins
based on the masses of their proteolytic fragments.
 Sequence Databases Contain the Amino Acid Sequences
of Proteins
 Protein sequence databases like SWISS-PROT and PIR
provide access to extensive protein sequence information.
 Mass spectrometry data
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o Can be used to search these databases to identify
and characterize proteins.
 The databases contain sequences from various species
and offer insights into protein evolution.
INTRODUCTION TO MASS SPECTROMETRY
 Mass spectrometry is a technique used to measure the
mass-to-charge ratio (m/z) of ions. It is a versatile and
sensitive analytical method that can be applied to a wide
range of compounds.
 Principle of ESI-MS:
o Electrospray ionization (ESI) is an ionization
technique used to create ions from sample
molecules in solution. In ESI, a sample solution is
sprayed through a fine capillary at the tip of a needle.
As the solvent evaporates, ions are formed from the
analyte molecules. ESI generates mostly multiply
charged ions.
 Ionization Process:
o In ESI, charged droplets are produced as the solvent
evaporates. These droplets carry charge, and as
they continue to evaporate, they form ions. The ions
produced are usually in the form of protonated
molecules (for example, [M+H]+ for small molecules
or [peptide+2H]2+ for peptides).
 Mass Analysis:
o Once ions are generated, they can be introduced
into a mass spectrometer. The mass spectrometer
separates ions based on their m/z ratio. The
resulting mass spectrum provides information about
the mass and charge of the ions, allowing the
determination of the molecular weight of the analyte.
 Applications:
o ESI-MS is widely used in proteomics, metabolomics,
and other fields to analyze complex mixtures of
biological molecules. It is used for protein
identification, characterization of small molecules,
and the study of non-covalent interactions.
TANDEM MASS SPECTROMETRY (MS/MS)
 Tandem mass spectrometry, often referred to as MS/MS, is
a more advanced mass spectrometric technique that
involves two consecutive stages of mass analysis. It
provides detailed structural information about the analytes.
 First Stage (MS1):
o In the first mass analysis, ions generated by ESI-MS
or another ionization method are separated based
on their m/z ratios. The ions of interest are then
selected for further analysis.
 Ion Activation:
o After the selection of specific ions, they undergo a
process called ion activation. This involves collision-
induced dissociation (CID), electron capture
dissociation (ECD), or other methods to induce
fragmentation of the selected ions. The goal is to
break the analyte into smaller pieces, generating
product ions.
 Second Stage (MS2):
o In the second stage of mass analysis, the product
ions are further analyzed using a mass
spectrometer. This results in a second mass
spectrum that provides information about the
fragments produced during ion activation.
 Structural Information:
o MS/MS provides valuable structural information
about the analyte. By examining the masses and
relative intensities of the product ions, researchers
can deduce the sequence, connectivity, and
structural features of the molecule. This is
particularly useful in the identification of peptides,
proteins, and the elucidation of unknown small
molecules.
 Applications:
o MS/MS is extensively used in proteomics,
metabolomics, pharmaceutical analysis, and
environmental chemistry. It plays a crucial role in
peptide sequencing, protein identification, and the
analysis of complex mixtures.
WHAT IS THE NATURE OF AMINO ACID
SEQUENCES?
 Amino Acids in Proteins:
o Proteins are essential biomolecules made up of long
chains of amino acids. Amino acids are the building
blocks of proteins, and the sequence in which they
are arranged within a protein determines the
protein's structure and, consequently, its function.
 Variety of Amino Acids:
o There are 20 standard amino acids that are
commonly found in proteins. Each of these amino
acids has a unique chemical structure, including a
central carbon atom, an amino group (NH2), a
carboxyl group (COOH), a hydrogen atom, and an
R-group, which is a variable side chain. It's the R-
group that differentiates one amino acid from
another.
 Amino Acid Frequency Analysis:
o Researchers often analyze large datasets of protein
sequences to determine how frequently each of the
20 amino acids appears within proteins. This
analysis provides valuable information about the
composition of proteins in various organisms and in
different protein families.
 Structural and Functional Insights:
o The relative frequencies of amino acids in proteins
have implications for the protein's three-dimensional
structure and its functional properties. For example:
 Amino acids with hydrophobic R-groups
o Tend to cluster in the interior of proteins to avoid
water (hydrophobic effect), contributing to protein
stability.
 Amino acids with polar R-groups
o Can be found on the protein's surface and may
participate in interactions with other molecules.
 The presence of specific amino acids at certain positions
within a protein can be critical for its function. For instance,
catalytic sites of enzymes often contain particular amino
acids that are essential for their activity.
EVOLUTIONARY INSIGHTS:
 Amino acid frequency analysis can also provide insights
into the evolutionary relationships between proteins.
o Proteins with similar functions or common ancestry
often share similarities in their amino acid
compositions. By comparing the frequencies of
amino acids in related proteins, scientists can infer
evolutionary relationships and trace the divergence
of protein families.
AMINO ACID SUBSTITUTION AND MUTATION
 Changes in the relative frequencies of amino acids in
proteins can be the result of mutations in the corresponding
genes. Some mutations may be neutral and not significantly
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affect protein function, while others can lead to functional o The process by which a gene is duplicated, leading
changes or even result in diseases. to the formation of related proteins.

COMPARATIVE STUDIES: APPARENTLY DIFFERENT PROTEINS SHARE

 Researchers often compare amino acid frequencies in COMMON ANCESTRY:
proteins from different species to study evolutionary  Hen Egg White Lysozyme:
adaptations. Such comparisons can reveal how organisms o A protein with antibacterial activity.
have developed specific proteins to suit their ecological  Human Milk α-Lactalbumin:
niches and lifestyles. o A protein involved in lactose synthesis.
 Tertiary Structure:
BIOINFORMATICS AND PROTEOMICS: o The three-dimensional arrangement of atoms in a
 Bioinformatics tools and databases are widely used for protein.
amino acid frequency analysis. This information is valuable  Structural Similarity:
in proteomics, which aims to understand the complete set o Proteins with different functions may have similar
of proteins in an organism and their functions. three-dimensional structures.
 The composition of amino acids in proteins reflects their
stability in aqueous environments. MUTANT PROTEIN:
 Unique amino acid sequences are critical for the distinct  Amino Acid Sequence:
functions of proteins. o Mutant proteins have variations in their amino acid
 Homologous proteins, both orthologous and paralogous, sequences due to gene mutations.
have related sequences and often perform similar  Neutral Mutations:
functions. o Mutations that do not significantly affect the protein's
 Homologous Proteins from Different Organisms Have function.
Homologous Amino Acid Sequences  Functional Consequences:
 Proteins with similar functions and structures in different o Mutations can lead to changes in the protein's
species are considered homologous. stability, solubility, and other properties.
 Orthologous proteins arise from a common ancestral gene
during evolution. CHEMICAL SYNTHESIS OF PEPTIDES AND
 Paralogous proteins arise through gene duplication. POLYPEPTIDES:
 Homology can be revealed through computer programs that  Orthogonal Synthesis:
align and compare protein sequences. o A synthetic strategy using distinct blocking groups to
protect functional groups while synthesizing
RELATED PROTEINS SHARE A COMMON peptides.
EVOLUTIONARY ORIGIN:  Peptide Bonds:
 Amino Acid Sequence Analysis: o Covalent bonds that link amino acids together.
o This method is used to compare proteins by  Peptide Synthesis:
examining the sequence of amino acids in their o The process of chemically creating peptides and
primary structure. polypeptides with defined sequences.
 Sequence Similarity:
o Proteins with related functions often display a high SOLID-PHASE METHODS IN PEPTIDE SYNTHESIS:
degree of sequence similarity, indicating a common  Solid-Phase Synthesis:
evolutionary ancestry. o A method in which the growing peptide chain is
covalently anchored to an insoluble resin, facilitating
OXYGEN-BINDING HEME PROTEINS: peptide synthesis.
 Myoglobin:  Automation:
o A protein that binds oxygen in muscles and consists o The cyclic process of solid-phase synthesis is often
of a single polypeptide chain. automated and controlled by computers.
 Hemoglobin:
o A tetrameric protein in erythrocytes consisting of two CONJUGATED PROTEINS:
α-chains and two β-chains, responsible for oxygen  Simple Proteins:
transport. o Proteins consisting of only amino acids.
 Paralogous Proteins:  Post-Translational Modifications:
o Proteins that share a common evolutionary origin o Chemical modifications to protein structure after
through gene duplication. synthesis.
 Sequence Homology:  Disulfide Linkage:
o The degree of similarity in amino acid sequence o A common post-translational modification involving
among related proteins. the formation of disulfide bonds.

SERINE PROTEASES: BIOLOGICAL FUNCTIONS OF PROTEINS:

 Trypsin, Chymotrypsin, and Elastase:  Proteome:
o These are serine proteases, a class of enzymes that o The entire set of proteins in an organism.
use specific serine residues for catalysis.  Binding Proteins:
 Thrombin: o Proteins that interact with other molecules (ligands)
o An essential serine protease involved in blood through noncovalent interactions.
clotting.  Transport Proteins:
 Gene Duplication:

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o Proteins that move substances across membranes
or deliver nutrients and waste products.
 Catalytic Proteins (Enzymes):
o Proteins that facilitate metabolic reactions.
 Regulatory Proteins:
o Proteins that control gene expression or respond to
environmental signals.
 Structural Proteins:
o Proteins that provide shape and support to cells and
structures.
 Protein–Protein Interaction:
o Interactions between proteins, which can involve
similar or different proteins.
LIGAND BINDING AND BINDING SITE:
 Dissociation Constant (KD):
o A measure of the strength of binding interactions
between a protein and its ligand.
 Binding Site:
o A specific region within a protein that interacts with
and binds to a ligand.
 Structural Complementarity:
o The matching of protein binding sites with ligand
structures for efficient binding.
SUMMARY
5.1 WHAT ARCHITECTURAL ARRANGEMENTS
CHARACTERIZE PROTEIN STRUCTURE?
 Proteins are generally categorized into three fundamental
structural classes: soluble, fibrous, and membrane-based
on their shape and solubility. In more detail, protein
structure is described in terms of a hierarchy of
organization:
 Primary (1°) structure:
o This refers to the protein's amino acid sequence.
 Secondary (2°) structure:
o This involves regular structural elements like helices
and sheets within the protein, created by hydrogen
bonds.
 Tertiary (3°) structure:
o This pertains to the folding of the polypeptide chain
in three-dimensional space.
 Quaternary (4°) structure:
o This involves the organization of subunits in
multimeric proteins.
 The three higher levels of protein structure are formed and
maintained through noncovalent interactions.
5.2 HOW ARE PROTEINS ISOLATED AND PURIFIED
FROM CELLS?
 Cells contain thousands of different proteins, and isolating
a specific protein from such complex mixtures can be
accomplished by exploiting two key physical properties:
size and electrical charge. Another approach is to use
affinity purification strategies that leverage the biological
function or specific recognition properties of a protein. A
typical protein purification strategy involves a series of
separation methods to obtain a pure preparation of the
desired protein.
5.3 HOW IS THE AMINO ACID ANALYSIS OF
PROTEINS PERFORMED?
 Acid treatment of a protein hydrolyzes all the peptide bonds,
resulting in a mixture of amino acids. Chromatographic
analysis of this hydrolysate reveals the amino acid
composition of the protein. Most proteins contain a variety
of the 20 common amino acids, with approximately 30%
being charged amino acids, 30% hydrophobic amino acids
(including aromatic amino acids), and the remaining being
polar, uncharged amino acids.
5.4 HOW IS THE PRIMARY STRUCTURE OF A
PROTEIN DETERMINED?
 The primary structure (amino acid sequence) of a protein
can be determined through various chemical and enzymatic
methods. Mass spectrometry can also be used. In chemical
and enzymatic protocols, a pure polypeptide chain with
disulfide linkages broken is used as the starting material.
Methods that identify the N-terminal and C-terminal
residues of the chain are employed to determine the amino
acids at the ends. The protein is then cleaved into smaller
fragments using enzymes such as trypsin or chymotrypsin
or chemical cleavage agents like cyanogen bromide. The
sequences of these products can be obtained through
Edman degradation. In mass spectrometry, an ionized
protein chain is broken into overlapping fragments, and
small differences in the masses of individual amino acids
are used to deduce the amino acid sequence.
5.5 WHAT IS THE NATURE OF AMINO ACID
SEQUENCES?
 Proteins have unique amino acid sequences, and sequence
similarity between proteins implies evolutionary
relatedness. Homologous proteins share sequence
similarity and structural resemblance, which can be used to
trace evolutionary histories. Sequence variation within a
protein is the result of mutations leading to amino acid
substitutions, and natural selection acts on these variants,
driving evolutionary change.
5.6 CAN POLYPEPTIDES BE SYNTHESIZED IN THE
LABORATORY?
 While challenging, it is possible to synthesize proteins in the
laboratory. Obstacles involve joining desired amino acids to
a growing chain using chemical methods that avoid side
reactions and undesired products. Techniques like solid-
state methods and orthogonal protection can circumvent
many of these issues, enabling the creation of polypeptide
chains with over 100 amino acid residues.
5.7 DO PROTEINS HAVE CHEMICAL GROUPS
OTHER THAN AMINO ACIDS?
 Many proteins undergo post-translational modifications to
certain amino acid side chains, which often regulate their
function. Additionally, proteins can be conjugated with
various other chemical components, such as
carbohydrates, lipids, nucleic acids, metal ions, and unique
structures like heme or flavin. These nonprotein
associations expand the physical and chemical properties
of proteins, creating a broader range of functional
possibilities.
5.8 WHAT ARE THE MANY BIOLOGICAL
FUNCTIONS OF PROTEINS?
 Proteins are key agents of biological function, often binding
various ligands, which is closely related to their function and
forms the basis for classification schemes. For instance:
o Transport proteins bind molecules for transportation.
o Enzymes bind reactants specific to the reactions
they catalyze.
o Regulatory proteins can either bind small molecules
that act as physiological or environmental cues (e.g.,
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hormone receptors) or bind to DNA, regulating gene

expression (e.g., transcription activators).
 Proteins typically interact with their ligands through
noncovalent interactions, and their specificity in ligand
binding is due to the complementary structure of the
protein's binding site with that of the ligand. Moreover, some
proteins function through interactions with other proteins,
forming the basis of many biological processes.

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