Treatment of a polypeptide with 2-mercaptoethanol yields two polypeptides:

1. Ala–Val–Cys–Arg–Thr–Gly–Cys–Lys–Asn–Phe–Leu

2. Tyr–Lys–Cys–Phe–Arg–His–Thr–Lys–Cys–Ser

Treatment of the intact polypeptide with trypsin yields fragments with the following amino acid compositions:

3. (Ala, Arg, Cys2, Ser, Val)

4. (Arg, Cys2, Gly, Lys, Thr, Phe)

5. (Asn, Leu, Phe)

6. (His, Lys, Thr)

7. (Lys, Tyr)

Indicate the positions of the disulfide bonds in the intact polypeptide.

The Cysteine residues contain a sulfur group in their side chain. Two polypeptide chains are linked when cysteine residue of one chain forms a disulfide bond with another cysteine residue of another chain. For determining the sequence of protein, these dislufide bonds must be broken to get two separate linear chains. 2–Mercaptoethanol is used to separate disulfide bonds formed between the peptide chains.

Trypsin is an endopeptidase isolated from Bovine pancreas.Only if the next residue is not Proline (Pro), Trypsin breaks the peptide bond the on the C side (carboxyl terminus) of positively charged amino acids Lysine (Lys) and Arginine (Arg).

When the given polypeptide is treated with 2 - mercaptoethanol, it yields two separate polypeptide chains. When the same polypeptide is treated with trypsin, it will yield several small fragments having the composition given in the question. This composition will only be obtained when disulfide bond is formed between the first Cys of 1 polypeptide and second Cys residue of 2 polypeptides; and between second Cys of 1 polypeptide and first Cys of 2 polypeptides.