Influence of high hydrostatic pressure on the proteolysis of β-lactoglobulin A by trypsin

Abstract

This work describes the effect of the hydrolysis time and pressure (0·1–400 MPa) on the proteolysis of β-lactoglobulin A (β-lg A) with trypsin, either conducting hydrolysis of β-lg under pressure or hydrolysing β-lg that was previously pressure treated. Pressurisation, before or during enzyme treatments, enhanced tryptic hydrolysis of β-lg. Trypsin degraded pressure-modified β-lg and pressure-induced β-lg aggregates, favouring proteolysis to the intermediate degradation products: (Val₁₅-Arg₄₀), (Val₄₁-Lys₆₉)S-S(Leu₁₄₉-Ile₁₆₂) and (Val₄₁-Lys₇₀)S-S(Leu₁₄₉-Ile₁₆₂). These were further cleaved at the later stages of proteolysis to yield: (Val₁₅-Tyr₂₀), (Ser₂₁-Arg₄₀), (Val₄₁-Tyr₆₀), (Trp₆₁-Lys₆₉)S-S(Leu₁₄₉-Ile₁₆₂) and (Trp₆₁-Lys₇₀)S-S(Leu₁₄₉-Ile₁₆₂). Particularly, in the tryptic hydrolysates of pre-pressurized β-lg, two other fragments linked by disulphide bonds: (Lys₁₀₁-Arg₁₂₄)S-S(Leu₁₄₉-Ile₁₆₂) and (Tyr₁₀₂-Arg₁₂₄)S-S(Leu₁₄₉-Ile₁₆₂), were found. These corresponded to rearrangement products induced by SH/SS exchange between the free thiol group of Cys₁₂₁ and Cys₁₆₀, that normally forms the disulphide bond Cys₆₆-Cys₁₆₀. In the light of these results, structural modifications of β-lg under high pressure are discussed.