PROTEINS, PEPTIDES, AND AMINO ACIDS

Composition and Structure

Unlike carbohydrates, proteins vary widely in composition, not only from one species to another but
also among the various tissues and cellular fluids within a given species. These differences in
composition result in different physical and chemical properties that are reflected in the diverse
biofunctions in which proteins participate.
The intimate roles these compounds play in the fundamental processes of tissue formation,
regeneration, and function makes this class of substances the primary component of all living matter,
and hence, the term protein (from the Greek, first). All proteins contain carbon, hydrogen, oxygen, and
nitrogen.
Nitrogen constitutes approximately 16% of most proteins, which leads to the rough factor of 6.25
generally employed to convert the amount of protein nitrogen found by analysis to the amount of total
protein. Other elements such as sulfur, phosphorus, iodine, copper, iron, and occasionally zinc may be
present.

The fundamental structural units of proteins are a-amino acids, about 20 of which prominently
participate in protein formation. These building-block molecules contain at least one carboxyl group and
one a-amino group, but differ in the structure of the remainder of the molecule. All except the simplest
one, glycine, are capable of existing in both D- and L- configurations with respect to their a-carbon, but
proteins contain only the L-enantiomers. The actual protein molecule consists of long-chain polymers
that have resulted from condensation of the amino acids, thus producing amide or peptide linkages:

RO

HRO H+ etc H

C-OH

HOH

In addition to the 20 standard amino acids several others of relatively rare occurrence have been
isolated from hydrolysates of some specialized types of proteins. All are derivatives of a standard amino
acid.

Hydroxylysine, the 5-hydroxy derivative of lysine, is present in collagen (as is hydroxyproline).

Desmosine and isodesmosine occur in the fibrous protein elastin. As noted below, desmosine can be
visualized as being formed from four lysine molecules with their side-chain moieties joined to form a
substituted pyridine ring.
Certain muscle proteins have been found to contain several e-N-methylated analogs of lysine and
histidine.
naturally occurring amino acids that are not found in proteins.

1.B-Alanine,
2. a-aminobutyric acid
3. homocysteine
4. homoserine
5.citrulline
6.ornithine
7.canavinine
8.djenkolic acid
9. ß-cyanoalanine

amino acids exist only in the free state

1. y-aminobutyric acid
2. a-aminoadipic acid
3. pipecolic acid
4. &-acetylornithine
5-Hydroxylysine
4-Hydroxyproline
Desmosine
E-N- Methyllysine
3-Methylhistidine
Table 26-6. Prominent Protein Amino Acids

Neutral Aliphatic

Glycine (Gly) CH2(NH2)COOH

aminoacetic acid

Alanine (Ala) CH3, CH(NH2)COOH

2-aminopropanoic acid

Serine (Ser) CH₂(OH)CH(NH₂)COOH

2-amino-3-hydroxypropanoic
acid

Threonine (Thr) CH3CH(OH)CH(NH₂)COOH

2-amino-3-hydroxybutanoic
acid

Valine (Val) CH3, CH(CH3)CH(NH2)COOH

2-amino-3-methylbutanoic acid

Leucine (Leu) CH(CH3CH(NH2)COOH

2-amino-4-methylpentanoic
acid

Isoleucine (Ile) CH, CH(NH2)COOH

2-amino-3-methylpentanoic
acid

Neutral Thioaliphatic
Cysteine (CySH) CH₂(SH)CH(NH₂COOH
2-amino-3-mercaptopropanoic
acid

Cystine (CyS-SCY) SCH₂CH(NH₂COOH

3,3-dithiodi(2-aminopropanoic
acid)

Methionine (Met) CH₂(SH3)CH2CH(NH₂)COOH

2-amino-4-(methylthio)butanoic
acid

Neutral Aromatic
Phenylalanine (Phe) 2-amino-3- CH2, CH(NH2)COOH
phenylpropanoic acid

Tyrosine (Tyr) -OH CH2, CH(NH2)COOH

2-amino-3-(p-hydroxyphenyl)
propanoic acid

Neutral Heterocyclic
Proline (Pro) 2- COOH
pyrrolidinecarboxylic acid

Hydroxyproline (Hyp) -COOH -OH

4-hydroxy-2-
pyrrolidinecarboxylic acid

Tryptophan (Trp) CH2,CH(NH2)COOH

a-aminoindole-3-propanoic
acid

Acidic
Aspartic Acid (Asp) HOOCCH2, CH(NH2)COOH
aminosuccinic acid

Glutamic Acid (Glu) HOOCCH₂CH₂CH(NH₂)COOH

2-aminoglutaric acid

Basic
Histidine (His) -CH2,CH(NH,)COOH
a-amino-4-imidazolepropanoic
acid

Lysine (Lys) CH2(NH2)CH2CH2CH2CH(NH2)COOH

2,6-diaminohexanoic acid

Arginine (Arg) NH2, C(=NHINHCH2,CH2,

CH2,CH(NH2)COOH
2-amino-5-guanidinopentanoic
acid
Proteins are macromolecules that differ from each other primarily in the number, type, and sequence of
amino acid residues present in the polymer chain. The number of amino acid molecules within proteins
ranges from perhaps as few as 30 and up to tens of thousands.

The term peptide is used in reference to very small hydrolytic fragments of proteins (generally having a
MW less than 10,000). They typically contain any where from 2 to possibly 20 or so amino acids joined
via amide linkages and are commonly subdivided into di-, tri-, etc, peptides according to the number of
amino acid residues they contain.

Collectively, higher molecular weight peptides are often termed as polypeptides.

Various individual peptides have been isolated from protein hydrolysates or synthesized, such as
oxytocin. With regard to classification or categorization, there is little distinction between peptides and
polypeptides, except that the latter usually refers to compounds that carry a number of amino acid
residues but usually do not involve a distinct upper limit of residues. For example, the polypeptide
hormone prolactin carries 199 residues.

The simplest naturally occurring peptides are the dipeptide penicillins and cephalosporins.

The MW of proteins may be determined by various methods, such as diffusion, sedimentation, viscosity,
x-ray analysis, light-scattering, ultracentrifugation, electron microscopy, and gel permeation. MW values
for common proteins range from about 10¹ to about 107; the value found for a given protein often
varies depending upon the determination method used.
Table 26-7. Amino Acid Composition of Selected Proteins

IUPAC ABBREVIATION

Ala

Arg Asp

Cys-Scy

Glu Gly

His

MILK

MIXED PROTEINS

GELATINS

Alanine

Arginine

Aspartic Acid Cystine

Glutamic Acid Glycine

Histisdine

Hydroxyproline Hyp
Isoleucine

Leucine

Lysine

Methionine Phenylalanine

Proline Serine

Threonine

Tyrosine

Tryptophan Valine

lle

Leu Lys

Met Phe

Pro Ser Thr

Tyr Trp

Val
SERUM ALBUMIN
GLOBULIN
HEMOGLOBLIN HORSE
INSULIN
CLOSTRIDIUM BOTULINIUM TOXIN

All values are in g/100 g of protein except those marked which are in g/16 g total nitrogen.

As for the elucidation of protein composition, two fundamental problems exist,

1.the quantitative assay of the individual amino acids and
2.the determination of the amino acid sequence in the chain.

Each is a highly specialized field of endeavor and relies upon modern techniques such as
1. selective adsorption (ion-exchange, paper, thin-layer, high-performance liquid, and gas-liquid
chromatography),
2. electrophoresis,
3. countercurrent distribution, and
4. isotope-dilution methods.
 In view of the diverse analytical methods employed, slight variations in reported values are expected
and often encountered in the literature.

With simple (nonconjugated) proteins, the total mass of the amino acids exceeds the mass of the
source protein because of the water that becomes fixed during hydrolytic cleavage of the peptide
linkages.

The precise sequence of amino acid residues is now known for a considerable number of proteins,
including insulin, ribonuclease, tobacco mosaic virus, and many of the hemoglobins,
immunoglobulins, and other specialized proteins.

Protein structure is typically divided into four levels:

1. Primary- The amino acid sequence, as determined by sequencing techniques.

2. Secondary-The folding of polypeptide chains into coiled structures as determined by X-ray
diffraction, optical rotatory dispersion, and electron photomicrography.
3. Tertiary-The arrangement of chains into specific layers and/or fibers.
4. Quaternary-The organization of many monomeric units, each displaying primary, secondary,
and tertiary structure, associated to form a quaternary structure.

A fifth level is believed to consist of aggregates of different proteins, each composed of the four
fundamental structural levels. These macromolecular complexes are believed to be involved in fatty acid
synthesis and electron transport.

Physical and Chemical Properties

A satisfactory practical classification of proteins based solely upon either composition or structure has
not been achieved, partly because of their wide diversity and partly because of in complete knowledge.
Classifications in terms of occurrence and function are encountered frequently in the literature but
these are designed for special purposes and usually do not embrace all proteins.

A classification based primarily upon physical and chemical properties such as solubility, coagulability,
conjugation, denaturation, and hydrolysis characteristics and having some practical utility has evolved
gradually over the years and is presented below.

Simple proteins are naturally occurring proteins that yield only a-amino acids or their derivatives upon
hydrolysis.
They may be of several types and include:
1. Albumins, which are soluble in water and coagulated by heat; examples include ovalbumin
in egg white and serum albumin in blood.
2. Globulins, which are insoluble in water but soluble in dilute salt solutions and coagulable by
heat; examples include serum globulin in blood.
3. Glutelins, which are insoluble in water or dilute salt solution but soluble in dilute acid and
alkali; examples include glutenin in wheat.
4. Prolamines, which are insoluble in neutral solutions but soluble in 80% alcohol; examples
include zein in corn and gliadin in wheat.
5. Albuminoids, which are dissolved only by boiling in strong acids; examples include keratins
in hair and horny tissue, elastins in tendons and arteries, and collagens in skin and tendons
 6. Histones, which are basic in reaction, soluble in water but insoluble in dilute ammonia, and
not easily heat-coagulable; examples include thymus histone and hemoglobin.
7. Protamines, which are strongly basic in reaction and soluble in water, dilute acid, and
ammonia; examples include salmin and sturin in fish sperm. They precipitate many other
proteins.

Conjugated proteins are proteins that are combined in nature with some nonprotein substance.
They are classified according to the nature of the prosthetic (nonprotein) group.

The classes, which are not mutually exclusive, include:

1. Phosphoproteins, which contain a phosphoric acid moiety as the prosthetic group; examples
include case in in milk and ovovitellin in egg yolk.
2. Nucleoproteins, which contain a nucleic acid as the prosthetic group; examples include nuclein
in cell nuclei.
3. Glycoproteins, which are simple proteins united to a carbohydrate group; examples include
mucins in vitreous humor and saliva.
4. Chromoproteins, which contain a colored prosthetic group; examples include hemoglobin in
blood and flavoproteins.
5. Lipoproteins, which contain lipid materials, such as sterols, fatty acids, or lecithin.
6. Metalloproteins, which contain a metal as the prosthetic group; examples include enzymes such
as tyrosinase, arginase, and xanthine oxidase.

In general, pure proteins are relatively odorless and tasteless and have varying colors. Many proteins
have been obtained in crystalline form, but unlike crystalline substances in general, this is not
necessarily evidence of homogeneity as some have been further resolved into two or more components
through chromatographic, electrophoretic, and other procedures.

Upon heating, proteins decompose with or without simultaneous liquefaction and emit the
characteristic odor of singed hair.

In their normal biological environment, they are highly hydrated.

Because proteins are polyelectrolyte macromolecules with multifunctional groups, they typically differ
greatly in their physical properties such as
1. solubilities in water,
2. salt solutions,
3. monohydric and polyhydric alcohols, and
4. dilute acids and bases.

Proteins often form colloidal solutions from which heat usually precipitates the protein in a
coagulated form.

Precipitation in an unaltered form is frequently accomplished, especially at their isoelectric

point, by means of salt solutions such as sodium chloride and ammonium sulfate or by diluted
ethanol.

Dilution with acetonitrile is often sufficient to precipitate protein from extracted serum
samples.
The exceptional vulnerability of proteins in general to chemical attack often requires careful control of
reaction conditions; nevertheless, their chemical characteristics are quite in accord with those to be
expected from the functional groups present.

Peptides are readily soluble in water, noncoagulable by heat, and are not precipitated by saturation with
ammonium sulfate.

Precipitates are formed with amino acids on the addition of various reagents such as heavy metal salts,
and certain acids such as picric, phosphotungstic, trichloroacetic, or sulfosalicylic acids.

In addition to the modern chromatographic, electrophoretic, and other procedures mentioned

previously, the advent of post-column-derivatization techniques in which peptides and amino acids are
made chromophoric by the use of such fluorescent derivatives as the
1. fluorescamine derivative,
2. the PTH amino acid derivatives,
3. the derivative formed by reaction in the orthophthaldehyde method, and
4. the dansyl and dapsyl derivatives

makes it possible to determine the concentration of individual amino acids and small peptides
in mixtures in the nanomole and picomole range.

In addition, the hydrolysis of proteins yields amino acids that, upon treatment with nitrous acid, liberate
nitrogen.

This reaction along with other techniques forms the basis of Van Slyke's nitrogen distribution method,
which has important uses in clinical chemistry.

Amino acids and the free amino groups in proteins react with ninhydrin resulting in either yellow, pink,
or violet color depending upon the amino acid.

The presence of peptide linkages can be shown by means of the Biuret test.

Numerous color tests are available for individual amino acids, including the
1. Ehrlich and Hopkins-Cole tests for tryptophan,
2. the Sak aguchi test for arginine,
3. the nitroprusside test for cystine and cysteine,
4. the Millon test for tyrosine,
5. the xanthoproteic test for tyrosine and phenylalanine,
6. the Pauly diazo test for histidine and tyrosine, and
7. the basic lead test for the sulfur-containing acids.

Occurrence and Uses

Proteins are synthesized by the ribosomes in the cytoplasm and especially those associated with
endoplasmic reticulum. Although proteins are present in all living matter, important differences in their
distribution are clearly evident.
In plants, for which the structural parts are essentially carbohydrate in nature, protein concentration is
usually very much higher in the seed than in any of other plant parts.

No similar gross variation is observed in the animal world, but different tissues vary considerably in the
approximate percentage of protein they contain (ie, skin-27%, skeletal muscle-21%, brain-11%, adipose
tissue-5%).

Insoluble proteins are usually isolated simply by removing contaminating material by means of a suitable
array of solvents.

Débridement is often facilitated through the appropriate use of enzymes.

Soluble proteins are usually obtained first as crude extracts in aqueous solutions and after subjecting the
solution to dialysis to remove contaminating solutes, the protein is obtained either through precipitation
by means of salt solutions or organic solvents or through lyophilization techniques.
When first isolated, proteins are frequently mixtures.

Separation into individual components was formerly accomplished only by means of tedious fractional
precipitation operations. Currently, it is achieved much more conveniently and completely through
chromatographic procedures using ion-exchange resins or various cellulose derivatives and preparative
HPLC.

In addition to their role in nutrition and as building blocks of proteins, the amino acids are precursors of
many important biomolecules, including various hormones, vitamins, coenzymes, alkaloids, and
porphyrins.

The aromatic amino acids are particularly versatile as precursors for many alkaloids, such as
morphine, codeine, and papaverine and

a number of hormones such as the thyroid hormone

1. thyroxine; the plant hormone,
2. indole acetic acid; and
3. an adrenal hormone, epinephrine.

Hormonal polypeptides are produced in the mammalian hypothalamus and are stored in the posterior
pituitary. Examples include the partially cyclic octapeptides oxytocin, vasopressin, argitocin, argipressin,
and lypressin.

The polypeptides ACTH (adrenocorticotropic hormone), lipotropin, prolactin, and soma totropin also
originate in the pituitary.

The hypothalamus produces the

1. polypeptide hormones or factors corticoliberin (CRF),
2. gonadorelin (GnRH),
3. protirelin (TRH), and
4. somatotropin releasing factor (GHF),
which are transported to the anterior pituitary.
Glutathione is a peptide hormone that is present in nearly all living cells.
 Other peptides (nonhormonal) in the hy pothalamus network are
1. neurotensin (anorexiant),
2. a tride capeptide, and
3. substance P, an undecapeptide.

The nerve tissue prevalent calcitonin gene-related peptide (CGRP) (37 residues) is 1000 times
more potent than acetylcholine or substance P. Cyclic polypeptides such as bacitracin and
polymixin from Bacillus sp. act as antibiotics, as do the penicillins and cephalosporins mentioned
previously.

LIPIDS

Lipids, also known as lipins or lipoids, are fat or fat-like substances that occur widely in plants (mainly
fruits and seeds) and animals (special deposits and in complex, active tissues such as the brain and liver).
They contain only carbon, hydrogen, and oxygen atoms except for complex lipids such as phospholipids.

Like the carbohydrates and proteins, the lipids constitute a very important group of organic substances
from a physiological standpoint. However, unlike the carbohydrates and proteins, the lipids comprise a
rather heterogeneous group of substances in terms of chemical composition.

In general, lipids are hydrophobic in nature, which is very important to their

physiological/pharmacological activities, and are soluble in solvents such as ether and chloroform and
insoluble in water.

They may be divided into the following classes according to their chemical structure:

1. Fixed Oils and Fats-Esters of glycerol and fatty acids. An example is olive oil. Fixed oils that are
solid at ordinary temperatures are commonly called fats. An example is lard.
2. Waxes-Esters of high-molecular-weight, monohydric alcohols and high-molecular-weight fatty
acids. An example is spermaceti.
3. Phospholipids (Phosphatides)-Esters consisting of glycerol in combination with fatty acids,
phosphoric acid, and certain nitrogenous compounds. Pharmaceutically, the most important
members of this group are the lecithins.
Prostaglandins-Essential fatty acids derived from prostanoic acid and having cyclic structures.

ALKALOIDS
- Are organic nitrogenous compounds w/c occur in plants, but some are found in animals
- They posses basic properties due to the presence of an amino nitrogen, marked physiologic
activity & often with a definite pharmacological action

Nomenclature : the name of the alkaloids are obtained in various ways :

- 1) From the generic name of the plant yielding them (hydrastine, atropine)
- 2) From the specific name of the plant yielding them (cocaine, belladonnine)
- 3) From the common name of the drug yielding them (ergotamine)
- 4) From their physiologic activity (emetine, morphine)
- 5) Occasionally from the discoverer (pelletierine)
• Forms of Alkaloids :
1) Free / pure form – basic form of alkaloid
- primary, secondary, tertiary amines
2) Salt form – synthetic, converts pure form to salt form w/c is done w/ the addition of acid
- quaternary amine
• Properties of Alkaloids :
1) In addition to carbon & hydrogen, they all contain nitrogen & generally oxygen
2) Most of the non-volatile alkaloids are solid, the volatile ones are mainly liquid
3) They are mainly crystallizable, though a few are amorphous
4) They are generally white though berberine is yellow & sanguinarine, itself is colorless yields
red salt
5) a. free / pure alkaloid are soluble in organic solvents but insoluble / sparingly soluble in water
b. alkaloidal salt are insoluble / sparingly soluble in organic solvents but soluble in water
6) Most of them are physiologically active some being extremely poisonous

7) a. pure alkaloid + acid = salt form

b. alkaloidal salt + base = pure alkaloid

8) They are precipitated by one / more of the ff reagents :

a. Mayer’s reagent – Mercuric potassium iodide

b. Marme’s reagent – Potassium cadnium iodide
c. Dragendorff’s reagent – Potassium bismuth iodide
d. Sonnenschein’s reagent – Phosphomolybdic acid
e. Wagner’s reagent – solution of iodine w/
potassium iodide
f. Scheibler’s reagent – Phosphotungstic acid
g. Gold chloride
h. Tannic acid
i. Picric acid
•
• Alkaloids are classified under the ff headings
I. OPIUM ALKALOIDS
Apomorphine Nalorphine
Codeine Naloxone
Hydrocodone Oxymorphone
Hydromorphone Papaverine
Morphine
II. CINCHONA ALKALOIDS
Quinidine Cinchonine
Quinine
III. TROPANE ALKALOIDS
Atropine Hyoscyamine
Benztropine Scopolamine
Cocaine Methscopolamine
Homatropine
 IV. XANTHINE ALKALOIDS
Aminophylline
Caffeine
Theophylline
V. ERGOT ALKALOIDS
Ergonovine Methylergonovine
Ergotamine Methysergide
VI. RAUWOLFIA ALKALOIDS
Reserpine Rescinnamine
VII. VERATRUM ALKALOID
VIII. VINCA ALKALOIDS
Vinblastine Vincaleukoblastine
Vincristine Leurocristine
IX. MISCELLANEOUS ALKALOIDS
Arecoline Physostigmine
Colchicine Pilocarpine
Emetine Tubocurarine
Ephedrine
•
• Identification tests :
1) Melting point
2) Specific rotation
3) Solubility in various solvents
4) Color reactions w/ specified reagents
5) Microscopic examination of crystals obtained by the action of suitable precipitants under
controlled conditions
•
• Method of Extraction :
1) Maceration
2) Percolation
3) Continuous Extraction