Question

1 (met) 2 (asp)    3 (gly) 4 (glu)    5 (trp)

The following pentapeptide MET-ASP-GLY-GLU-TRP is initially treated with cyanogen Bromide, and subsequently treated with Chymotrypsin. Based on this information, answer the following: Identify the carboxyl and amino terminus of the pentapeptide before treatment and indicate if terminal side chain groups are ionizable? If applicable, indicate which reagents result in chemical cleavage or enzymatic cleavage. Biochemical approaches can be used to determine the differences in the peptide before and after treatment. Indicate two biochemical approaches and what is being analyzed using this approach. Indicate any free amino acids and/or peptide fragments that result after cyanogen bromide treatment. If applicable, indicate any free amino acids and/or peptide fragment sequences that result after cyanogen bromide treatment and subsequent treatment with chymotrypsin.

Answer 1

Ans. #1. C-terminal residue = Trp

            N-terminal residue =Met

            The side chains of N-ter as well as C-ter residues are NOT ionizable.

#2. The following reagents and enzymes are used here for cleavage of the peptide chain-

CNBr- the chemical reagent

Chymotrypsin – Enzyme

#3. CNBr treatment is used to detect the presence of methionine residues in the peptide. Cyanogen bromide hydrolyzes the peptide at C-terminus of methionine in the target peptide bond.

# Chymotrypsin digests the peptide at the COO- terminus of a Tyr, Trp and Phe in the target peptide bond.

Chymotrypsin does not hydrolyze the peptide chain if the target residue is at C-terminus. Since Trp residue is at C_ter, the tetrapeptide produced after CNBr treatment won’t be acted upon by chymotrypsin.

Chymotrypsin digestion can be used, in combination of other enzymes and reagents, to determine the amino acid sequence of a polypeptide chain or protein.

#4. CNBr Treatment – Free amino acid produced is Met.

Chymotrypsin treatment : No effect.