Influence of buffer type and concentration on the peptide composition of trypsin hydrolysates of β-lactoglobulin

Proteins acquire conformation in aqueous media due to the influence of the buffer salt composition and concentration. Such influence may have impact on the enzyme–substrate interaction and somehow steer the enzyme attack properties, leading to release of dissimilar products. Our group has sought to investigate the influence of the hydrolysis environment on the trypsinolysis of a model protein, β-lactoglobulin (β-Lg). This work was aimed at investigating the effect of different buffers and their concentrations on the trypsinolysis patterns of β-Lg. The traditional NaOH-buffered water, in comparison to Tris–HCl and potassium-phosphate buffer at 62.5 mM–1.0 M were used at pH 8.5 for the pH drop and pH 7.8 for the hydrolysis. Bovine trypsin (EC 3.4.21.4) was used at an enzyme-to-substrate ratio of 1%. The samples were analysed for mass composition, using LC-ESI-TOF/MS and MALDI-TOF/MS for monitoring time-dependence of peptide evolution. In all buffer types and concentrations, peptides f(1–8), f(15–40, f(125–138) and f(142–148) were detected, implicating ease of hydrolysis of the terminal regions of β-Lg. A peptide from f(9–14), with sequence Gly-Leu-Asp-Ile-Gln-Lys, was detected at >0.5 M Tris–HCl only, while peptide f(71–75) was unique to <125 mM Tris–HCl and >250 mM potassium-phosphate buffer. Hydrolysis under buffer produced trypsin-specific peptides, numerous chymotrypsin-like non-specific peptides but no disulphide-linked peptides. Trypsinolysis shifted to the N-terminal region of lysine under some conditions. Hydrolysis under buffer holds potential for the avoidance of some peptides with undesirable characteristics while preserving a diversity of different peptides with possible bioactive properties.