Quantitative peptide release kinetics to describe the effect of pH on pepsin preference

Abstract

The preference of pepsin to hydrolyse certain peptide bonds is typically determined by counting peptides after hydrolysis, without considering concentrations and kinetics. In this study, peptide release was quantified to describe proteolysis by pepsin. The aim was to investigate whether pH affects individual hydrolysis rates of peptide bonds. α-Lactalbumin was hydrolysed by porcine pepsin systematically at pH 1–5 and peptides were identified and quantified with UPLC-PDA-MS at eight time points. Apparent pH-based differences in specificity were caused by differences in total hydrolysis rate but the relative hydrolysis rates of cleavage sites were generally independent of pH. Previous statements of pepsin preference for amino acids in the P3-P3′ positions withstand when considering the hydrolysis rates of cleavage sites and was pH independent. Despite the a-specificity of pepsin, many bonds were not or slowly hydrolysed, some cleavage sites became more accessible during hydrolysis (demasking) and some became less accessible (masking).