Learn about::-  1. Definition of Peptides 2. Primary Structure of Peptides 3. Determination of the Primary Structure 4. Synthesis 5. Physiologically Active.

Contents:

  1. Definition of Peptides
  2. Primary Structure of Peptides
  3. Determination of the Primary Structure of Peptides
  4. Synthesis of Peptides
  5. Physiologically Active Peptides

1. Definition of Peptides:

A peptide consists of two or more amino acids linked by a peptide bond formed between the carboxyl group of one amino acid and the amino group of another with the removal of one mole of water during a peptide bond.

Polypeptides are formed by long peptide chains containing large numbers of peptide bonds. Polymers of up to 100 amino acids are termed polypeptides and those with more than 100 are generally termed proteins.

2. Primary Structure of Peptides:

The sequence of amino acids in a polypep­tide is referred to as its primary structure. Chemical methods are required to determine the primary struc­ture.

Physical technics such as X-ray crystallogra­phy are employed for higher order of protein struc­ture.

The N-terminal (amino terminal) amino acid is always shown at the left and the C-terminal (car­boxyl terminal) amino acid at the right of the polypeptide chain.

Even small changes in the primary structure of proteins may produce remarkable physiologic effects.

Substitution of a single amino acid for an­other in a sequence of 100 or more amino acids may abolish biologic activity with serious conse­quences (e.g., Sickle cell disease).

The biochemical basis for many inherited metabolic diseases are known owing to the introduction of new chemical and physical methods to determine protein struc­ture.

3. Determination of the Primary Structure of Peptides:

A. Peptides are purified before Analysis:

i. The classic purification procedures in­clude precipitation with varying salt con­centrations (ammonium or sodium sulfate) or solvents (acetone or ethanol), differen­tial centrifugation, gel filtration and elec­trophoresis.

2. Selective absorption and elution of pro­teins from the cellulose anion exchanger diethylaminoethyl (DEAE) cellulose and the cation exchanger carboxymethyl cel­lulose (CMC) have also been extremely successful for excessive and rapid purifi­cation of protein,

B. Determination of Amino Acid Composition:

i. The peptide bond linking amino acid residues are first broken by hydrolysis.

ii. The liberated amino acids are then sepa­rated and identified by HPLC or ion ex­change chromatography.

C. Determination of Sequence of Polypeptide by Sanger:

i. Sanger’s approach was first to separate the two polypeptide chains A and B of insu­lin and then to convert them by specific enzymatic cleavage into smaller peptides that contained regions of overlapping se­quence.

Using 1 fluoro-2, 4-dinitrobenzene, he then removed and identified, one at a time, the amino terminal amino acid residues of these peptides—thus he was able to deduce an unambiguous primary structure for both the A and the B chains.

D. Primary Structures are determined by the Automated Edman Technique:

i. Since many proteins consist of more than one polypeptide chain associated by non-covalent forces or disulfide bridges. The first step to dissociate and separate individual polypeptide chains.

ii. Denaturing agents (urea, guanidine hydro­chloride) disrupt hydrogen bonds and dis­sociate non-covalently associated poly peptides.

iii. Oxidizing and reducing agents disrupt disulfide bridges. Polypeptides are then separated by chromatography.

iv. In this automated technique, the Edman reagent is phenyl isothiocyanate. The re­action sequence releases the amino termi­nal amino acid as a phenylthiohydantoin derivative which is then identified by HPLC.

v. The next amino acid in sequence is then derivatized and removed, and then the process is repeated.

vi. The Edman reactions take place either in a thin film on the wall of a spinning cup reaction chamber or on a solid matrix to which the carboxyl terminal of the pep­tide has been covalently coupled.

E. Large Polypeptides are cleared before being Sequenced:

i. Automated sequencing instruments oper­ate most efficiently on polypeptides of 20-60 residues long. Specific and com­plete cleavage at comparatively rare sites thus is desirable.

The following reagents meet this requirement:

(a) Cyanogen Bromide (CNBr)

(b) Trypsin.

(c) O-Iodosobenzene.

(d) Hydroxylamine.

(e) Protease V8.

(f) Mild acid hydrolysis.

4. Synthesis of Peptides:

Peptides may be synthesized by a reaction between an activated carboxyl group such as an acid chlo­ride of one amino acid and the amino group of an­other, e.g., cysteine acid chloride and lysine.

After the peptide bond is formed, the block­ing group is removed, leaving the desired peptide.

5. Physiologically Active Peptides:

i. In some cases, they are the basis of prod­ucts of protein.

ii. In others, they may be hormones, antibi­otics, precursors of bacterial cell walls, or even potent poisons.

iii. The widely distributed tripeptide glutath­ione is required for the action of several enzymes including insulin. Glutathione reductase functions either in insulin deg­radation or in the formation of disulfide bonds.

iv. Other naturally occurring important peptides are Bradykinin and Kallidin which are smooth muscle hypotensive agents are liberated from specific plasma proteins by treatment with snake venom.

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