Tryptophan Fluorescence and Time-Lag Hydrolysis of Peptide Bonds during Degradation of β-Lactoglobulin by Trypsin

Mikhail M Vorob’Ev

doi:10.3390/catal10121368

Abstract

The opening of protein globules and corresponding exposure of their internal peptide bonds, the so-called demasking effect, is required for successful hydrolysis of peptide bonds by proteases. Under the proteolytic action of trypsin on β-lactoglobulin (β-LG), the evolution of tryptophan fluorescence spectra showed that the demasking process consists of two stages with different demasking rate constants for each stage. It was found that the ratio of these constants depends on the concentration of trypsin and changes are approximately threefold when the concentration of trypsin changes in the range of 0.3–15 mg/L. Simulation of hydrolysis taking into account the demasking effect demonstrated how the apparent first-order rate constants obtained experimentally are related to the true hydrolysis rate constants and demasking parameters. The lag phase in the kinetic curves corresponding to the hydrolysis of various peptide bonds in β-LG was also analyzed. The increased lag times indicated sites that are hydrolyzed by a two-stage demasking mechanism.

Highlights

Modeling of enzymatic hydrolysis of proteins, known as proteolysis, has always been given considerable attention due to the importance of proteolysis processes in biology, biotechnology and food science [1,2]
We suggested that demasking manifests itself differently at the initial stage of hydrolysis, when demasking differently stage ofstage hydrolysis, when theTwo protein globule is the proteinmanifests globule isitself destroyed, andat atthe theinitial intermediate of proteolysis
Here that thebonds hydrolysis of peptide consists of several stages and depends j with this approach, here we propose that the hydrolysis of peptide bonds consists of several stages both on the parameters of demasking and on the true hydrolysis rate constants k

Summary

Introduction

Modeling of enzymatic hydrolysis of proteins, known as proteolysis, has always been given considerable attention due to the importance of proteolysis processes in biology, biotechnology and food science [1,2]. As the protein globule breaks down, intrinsic peptide bonds open up for the enzyme, so that they can only be hydrolyzed from some time of proteolysis [3,4]. This process of opening peptide bonds, called demasking, often limits the hydrolysis of peptide bonds and is an important component of proteolysis [5]. Inhibition of the enzyme by proteolysis products and even its irreversible inactivation determines the overall kinetics of proteolysis

Methods

Results

Conclusion