Peptides

Peptide

Compound formed by linking amino acids through peptide bond (covalent linking between a-carboxyl group of an amino acid and a-amino group of adjacent amino acid) which have unbranched chains.

1. Naturally occurring

2. Generated by chemical hydrolysis (6M HCL)

3. Generated by enzymatic hydrolysis

Peptide bond

1. 4 atoms of peptide bond and the 2 attached a-C are all in the same spatial plane (rigid and planar).

2. The carbonyl and imino bond dipoles are in opposite directions relative to the C-N bond axis = always in Trans configuration which is favored. Except proline which favors cis

3. There is no freedom of rotation about the bond between the carbonyl carbon atom and the imino N atom. The bond has partial double bond as a result of 1.32 Å which is between single bond (1.49Å) and double bond (1.27Å). Linkage has some double bond and single bond characteristics.

4. Rotation around the C-N peptide bond axis is severely restricted because of its double bond nature. Due to free rotation around majority of bonds along the backbone the peptide chain can easily change its orientation

Naturally occurring peptides

1. Carnosine (Ɓ-alanylhistidine): Dipeptide found in muscle tissue of vertebrates including human muscles. May function as a pH buffering agent in muscle cells

2. Glutathione (ƴ-glutamylcysteinylglycine): Transports amino acids across cell membrane. Scavenger of toxic oxidizing agents. (Note; N-terminal glutamine acid is denoted at ƴ-glutamyl because the R group ƴ-COOH participates in peptide bond linkage)

3. Oxytocin: stimulates contractions of uterine muscles in pregnant females and ejection of milk from mammary glands in lactating females.

   Vasopressin: Produces potent antidiuretic effects reabsorption of H2O by the kidney

4. Gramocidin-S: Cyclic decapeptide antibiotic, produced by bacteria. Contains 2 non-protein amino acids. Used as an antibiotics to treat bacterial eye infection.

Electrolyte behavior of peptides

Free terminal COOH group of the peptide has a pKa=3.

Free terminal amino group has a pKa=8.

The pKa values on ionisable R groups are the same as those displayed by R groups of individual AA's.

Chemical reactions

1. Free N-terminal amino group of peptides undergoes the same reactions as those given by a-NH2 groups of free AA's: -FDNB reaction. -Dansyl Chloride. -Edman Reagent

2. C-terminal COOH group undergoes the same reactions as those given by the a-COOH groups of AA's: -Reduction with LiBH4 or NaBH4. -Hydrazinolysis

3. Biuret reactions is the only reaction specific for peptides and proteins. Alkaline CuSO4 reacts with compounds containing two or more peptide bonds to give purple colour at 570nm.

Amino acid composition

Peptide/protein is hydrolyzed to determine its constituent AA's. The relative amount of each AA may be determined by chromatography techniques.

1. Acid hydrolysis: peptide is heated with excess 6M HCL at 100° for 10-24 hours. Excess HCL removed and aa remain as their HCL salts. Advantage: little to no racemization of AAs. Disadvantage: Trp destroyed and Gln converted to Glu and Apn converted to Asp.

2. Alkaline hydrolysis: advantage of boiling with alkali (NaOH) is the Trp remains stable. Disadvantages is racemization and Cys, Ser and Thŕ are destroyed

Incomplete hydrolysis of proteins to produce peptides

Polypeptide is large which makes it hard to study therefore when studied it’s fragmented this is achieved by using enzymes known as endopeptidases which hydrolyzed specific internal peptide bonds in the central sections of polypeptide chains.

Identification of amino terminal

1. Chemical analysis: (Sangers reaction, Dansyl Chloride reaction, Emmanuel Reaction) Only the N-terminal residue will react. Thereafter the peptide chain is hydrolyzed completely. The individual aa are analyzed by chromatography and the aa derivative resulting from any one of the reactions above is derived from amino terminal residues.

2. Enzymatic analysis: Exopeptudase leucine aminopeptidase is employed. It removes the N-terminal residues and leaves the rest of the polypeptide chain intact. The released amino acid may be analyzed and identified. Disadvantage is reaction is progressive.

Edman Degradation procedure

Permits removal and ID of one residue at a time from the N-terminus of a polypeptide. Involves treating a polypeptide with PITC at pH9.

Identification of carboxyl terminal amino acids residue of peptide

1. Chemical analysis: (Reduction with LiBH4/NaBH4, Hydrazinolysis) Only the C-terminal residues will react. Thereafter the peptide chain is hydrolyzed completely.

2. Enzymatic analysis: Exopeptidase carboxypeptidase A,B ir C is employed. It removes the C-terminal residues and leaves the rest of the polypeptide chain intact.