Comparison of Protein Hydrolysis Catalyzed by Bovine, Porcine, and Human Trypsins
Deng, Yuxi ; Gruppen, Harry ; Wierenga, Peter A. - \ 2018
Journal of Agricultural and Food Chemistry 66 (2018)16. - ISSN 0021-8561 - p. 4219 - 4232.
LC-MS - peptide release kinetics - protein digestibility - secondary specificity - tryptic hydrolysis
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 (DHmax,theo). This is in contrast with experiments. Using α-lactalbumin and β-casein, this study aims to reveal if the differences among experimental DHmax (DHmax,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 DHmax predictions based on the secondary specificity were 4 times closer to the DHmax,exp than the predictions based on trypsin specificity alone (DHmax,theo). Proposed preliminary relations between binding sites and trypsin secondary specificity allow DHmax,exp estimations of tryptic hydrolysis of other proteins.
Introducing enzyme selectivity: a quantitative parameter to describe enzymatic protein hydrolysis
Butré, C.I. ; Sforza, S. ; Gruppen, H. ; Wierenga, P.A. - \ 2014
Analytical and Bioanalytical Chemistry 406 (2014)24. - ISSN 1618-2642 - p. 5827 - 5841.
bovine beta-lactoglobulin - tryptic hydrolysis - substrate-specificity - mass-spectrometry - release kinetics - casein - peptides - identification - ph - proteases
Enzyme selectivity is introduced as a quantitative parameter to describe the rate at which individual cleavage sites in a protein substrate are hydrolyzed relative to other cleavage sites. Whey protein isolate was hydrolyzed by Bacillus licheniformis protease, which is highly specific for Glu and Asp residues. The molar concentration of all peptides (58) from ß-lactoglobulin formed during hydrolysis was determined from the UV214 signal. The quality of identification and quantification of the peptides were described by newly defined parameters: the peptide sequence coverage (on average 94 %) and the molar sequence coverage (on average 75 %). The selectivity was calculated from the rate of hydrolysis of each cleavage site, and showed differences of up to a factor of 5,000. The ability to quantitatively discriminate the enzyme preference towards individual cleavage sites is considered essential to the understanding of enzymatic protein hydrolysis.