Amino Acids, Peptides and Proteins - Dévényi T., Gergely J. 1976

Hydrolysis of proteins and peptides
Hydrolysis of proteins and peptides by chymotrypsin

Principle of the method. Chymotrypsin cleaves peptide bonds formed by the carboxyl groups of aromatic Amino Acids.

PROCEDURE

To a 1% solution of the substrate (protein) to be hydrolyzed, crystalline chymotrypsin is added at a ratio of 1/30 (relative to The amount of protein). The mixture is placed in an autotitrator and hydrolyzed under a nitrogen atmosphere with gentle stirring, maintaining pH 8.0 by the automatic addition of 0.1 N NaOH solution. Hydrolysis is continued until the consumption of the alkali solution ceases. This typically requires 4–8 h, after which the enzyme is inactivated by heating the reaction mixture in a boiling Water bath for 10 min. The precipitate is removed by filtration, and the filtrate is lyophilized.

NOTES

1. Chymotrypsin is much less specific than Trypsin. Selective Cleavage of peptide bonds involving the carboxyl groups of aromatic amino acids occurs only during short-term hydrolysis. Prolonged hydrolysis leads to the cleavage of other peptide bonds as well, such as those formed by leucine, Histidine, and glutamine residues.

2. Tyr-Pro and Phe-Pro bonds are highly resistant to chymotrypsin action, while bonds like Tyr-Tyr are partially resistant.