Comparison of Protein Hydrolysis Catalyzed by Bovine, Porcine, and Human Trypsins

[Image: see text] Based on trypsin specificity (for lysines and arginines), trypsins from different sources are expected to hydrolyze a given protein to the same theoretical maximum degree of hydrolysis (DH(max,theo)). This is in contrast with experiments. Using α-lactalbumin and β-casein, this study aims to reveal if the differences among experimental DH(max) (DH(max,exp)) by bovine, porcine, and human trypsins are due to their secondary specificity. Peptide analysis showed that ∼78% of all the cleavage sites were efficiently hydrolyzed by porcine trypsin, and ∼47 and ∼53% were efficiently hydrolyzed by bovine and human trypsins, respectively. These differences were explained by the enzyme secondary specificity, that is, their sensitivities to the amino acids around the cleavage sites. The DH(max) predictions based on the secondary specificity were 4 times closer to the DH(max,exp) than the predictions based on trypsin specificity alone (DH(max,theo)). Proposed preliminary relations between binding sites and trypsin secondary specificity allow DH(max,exp) estimations of tryptic hydrolysis of other proteins.