Question

The protein pictured is bovine insulin. Determine the residue number and the size of the fragments that would be generated upon treatment with following proteases (1) trypsin, (2), chymotrypsin and (3) BrCN; (a) without DTT and (b) with DTT. (Where DTT breaks disulfide bonds).

Answer 1

Bovine insulin's protein structure is affected by cleavage with trypsin, chymotrypsin, and BrCN. Without DTT, larger fragments are formed due to intact disulfide bonds; with DTT, smaller fragments result from the cleavage of the now-separated A and B chains.

Step-by-step explanation:

The peptide hormone bovine insulin consists of two chains, A and B, which are connected by disulfide bonds. Treatment with various proteases leads to cleavage at specific residues, producing peptide fragments.

(1) Trypsin cleaves at the C-terminal side of lysine and arginine residues. Without DTT, the disulfide bonds remain intact, and larger fragments incorporating both A and B chains would result. With DTT, which reduces disulfide bonds, the chains would be separated, and each chain would then be cleaved at lysine and arginine residues, resulting in smaller fragments.

(2) Chymotrypsin preferentially cleaves at the carboxyl groups of aromatic amino acids (phenylalanine, tryptophan, and tyrosine). The resulting fragments would thus depend on the locations of these amino acids within the insulin chains. With DTT, the chains would be cleaved into smaller fragments, as the disulfide bonds would be broken.

(3) BrCN (2-bromo-2-methylpropionyl bromide) significantly increases the size of peptide fragments generated upon treatment. Without DTT, BrCN would generate larger fragments compared to with DTT, where the disulfide bonds would be broken prior to cleavage.

Answer 2

The residue number and size of the fragments that would be generated upon treatment with proteases should be as follows:

Step-by-step explanation:

1. Without DTT:

• Trypsin 1, 7 and 43 amino acids.

• Chymotrypsin 1, 1, 1, 4, 5, 10, 29.

• BrCN 51

2. With DTT:

• Trypsin 1,7, 21 and 22 amino acids
• Chymotrypsin 1,1, 1, 2, 4, 5, 8, 14, 15.
• BrCN 21 and 30 amino acids