Question

The following reagents are often used in protein chemistry: CNBr, Urea, Mercaptoethanol, Chymotrypsin, Trypsin, Performic acid, 6 N HCl, and Phenyl isothiocyanate. Which one is the best suited for accomplishing each of the following tasks?

a) Determination of the amino acid sequence of a small peptide.

b) Reversible denaturation of a protein devoid of disulfide bonds. Which additional reagent would you need if disulfide bonds were present?

c) Hydrolysis of peptide bonds on the carboxyl side of aromatic residues.

d) Cleavage of peptide bonds on the carboxyl side of methionines.

e) Hydrolysis of peptide bonds on the carboxyl side of lysine and arginine residues.

Answer 1

a) Phenyl isothiocyanate reagent is used for the determination of the amino acid sequence of a small peptide.

Explanation: In alkaline medium, phenyl isothiocyanate reacts with the N-terminal amino group and forms a cyclic phenylthiocarbamoyl derivative. In acidic medium, this derivative of the terminal amino acid will be cleaved as a thiazolinone derivative.

b) Urea, mercaptoethanol

Explanation: Urea is responsible for the reversible denaturation of a protein devoid of disulfide bonds, whereas mercaptoethanol is needed to reduce disulfides.

c) Chymotrypsin

Explanation: Chymotrypsin specifically cleaves the C-terminal side of aromatic amino acid residues.

d) CNBr

Explanation: Cyanogen bromide specifically cleaves the C-terminal side of methionine residues.

e) Trypsin

Explanation: Trypsin specifically cleaves the C-terminal side of lysine and arginine residues.