You wish to sequence the light chain of a protease inhibitor from the Brassica nigra plant. Cleavage of the light chain by trypsin and chymotrypsin yields the following fragments. What is the sequence of the light chain?

Chymotrypsin

1. Leu–His–Lys–Gln–Ala–Asn–Gln–Ser–Gly–Gly–Gly–Pro–Ser

2. Gln–Gln–Ala–Gln–His–Leu–Arg–Ala–Cys–Gln–Gln–Trp

3. Arg–Ile–Pro–Lys–Cys–Arg–Lys–Phe

Trypsin

4. Arg

5. Ala–Cys–Gln–Gln–Trp–Leu–His–Lys

6. Cys–Arg

7. Gln–Ala–Asn–Gln–Ser–Gly–Gly–Gly–Pro–Ser

8. Phe–Gln–Gln–Ala–Gln–His–Leu–Arg

9. Ile–Pro–Lys

10. Lys

The sequence of a protein is determined by breaking the intact polypeptide into smaller fragments of amino acids. This process is repeated twice using 2 different endopeptidases—enzymes that break the peptide bond present between the amino acids, to produce 2 different sets of fragments of the same polypeptide. These fragments are sequenced and then overlapped to determine the whole sequence of the polypeptide.

Trypsin and Chymotrypsin are endopeptidases which are isolated from Bovine pancreas. Only when the next amino acid residue is not Proline (Pro), Trypsin breaks the peptide bond on the C side (carboxyl terminus) of positively charged amino acids Lysine (Lys), and Arginine (Arg).

Only when the next residue is not Proline, Chymotrypsin cleaves peptide bonds on the C side (carboxyl terminus) of bulky hydrophobic residues which are Tyrosine (Tyr), Phenylalanine (Phe), and Tryptophan (Trp).

When the fragments obtained of light chain of protease inhibitor treated with trypsin and chymotrypsin are overlapped, they give the whole sequence of the light chain.

Thus, the sequence of the protease inhibitors light chain is

Arg – Ile – Pro – Lys – Cys – Arg – Lys – Phe – Gln – Gln – Ala – Gln – His – Leu – Arg – Ala – Cys – Gln – Gln – Trp Leu – His – Lys – Gln – Ala – Asn – Gln – Ser – Gly – Gly – Gly – Pro – Ser.