Question

Separate cleavage reactions of a polypeptide by CNBr and chymotrypsin yield fragments with the following amino acid sequences. What is the sequence of the intact polypeptide?

CNBr treatment

1. Arg–Ala–Tyr–Gly–Asn

2. Leu–Phe–Met

3. Asp–Met

Chymotrypsin

4. Met–Arg–Ala–Tyr

5. Asp–Met–Leu–Phe

6. Gly–Asn

Short answer

The intact peptide has the following sequence:

Asp – Met – Leu – Phe – Met – Arg – Ala – Tyr – Gly – Asn

Step-by-step solution

Protein sequencing

A protein must first be broken down into smaller fragments for it to be sequenced. To break the peptide into smaller fragments, endopeptidases such as pepsin, trypsin, and chymotrypsin are used. Two separate peptidases are used to break the peptide, yielding two different sets of fragments. The amino acid sequences of each fragment are then determined. These sequenced fragments are overlapped to determine the entire polypeptide sequence.

Cleavage by Chymotrypsin and Cyanogen bromide

Chymotrypsin is an endopeptidase purified from Bovine pancreas. Only if the next residue is not Proline (Pro), Chymotrypsin cleaves peptide bonds at the carboxyl terminus (on the C side) of bulky hydrophobic residues Tryptophan (Trp), Phenylalanine (Phe), and Tyrosine (Tyr).

There are many reagents that also promote cleavage of peptide bond at specific sites. Cyanogen Bromide (CNBr) is one such reagent. It cleaves the peptide bond on carboxy terminal of Methionine (Met).

Explanation

The fragments obtained by cleavage with CNBr and chymotrypsin are overlapped.

Thus, the intact polypeptide has the sequence:

Asp – Met – Leu – Phe – Met – Arg – Ala-Tyr – Gly – Asn.