Kinetic modeling of demasking and hydrolysis of peptide bonds during proteolysis of ß-lactoglobulin by trypsin.

Proteolysis of ß-lactoglobulin by trypsin was studied with fluorescence spectroscopy and an empirical exponential model was engaged to describe the peptide bond hydrolysis kinetics. The shift in the fluorescence maximum of tryptophan residues, from 342 to 352 nm, in the course of ß-lactoglobulin degradation was used as an indicator of the transition of masked peptide bonds to the demasked ones, which were accessible for the enzyme action. A simple equation with only two parameters was suggested to link together the degree of demasking of peptide bonds and the degree of their hydrolysis, allowing the kinetic description of proteolysis.

[Classification of enterprises according to degree of accident and occupational disease risk]. / O klassifikatsii predpriiatii po stepeni riska neschastnykh sluchaev i profzabolevanii.

The authors suggested approach to classification of enterprises and economy branches according to occupational conditions. Suggestion is to classify by means of quantitative methods based on claster analysis.

Kinetics of beta-casein hydrolysis by wild-type and engineered trypsin.

Apparent rate constants of tryptic hydrolysis of amide bonds containing Arg and Lys residues in beta-casein were determined by the analysis of kinetics of accumulation of 17 major peptide components revealed by high performance liquid chromatography. When studying pH influence on Arg/Lys bond cleavage preference, averaged rate constants over several Arg&bond;X and Lys&bond;X bonds were used for analysis of kinetics of wild-type trypsin, K188H, K188F, K188Y, K188W, and of K188D/D189K mutants. The pK(a1) value of 6.5 was found for all studied trypsins. For wild-type trypsin and its K188D/D189K mutant, pK(a2) was found to be 10. The lowest among studied engineered trypsins pK(a2) = 9.3 was determined for K188Y mutant. Considerable preference for the cleavage of Arg over Lys containing peptide bonds was demonstrated for all trypsins with engineered S2 site except for K188H and K188F. The comparison of individual rate constants for various bonds showed that during the hydrolysis by wild-type trypsin, the probabilities of splitting depend on secondary specificity and local hydrophobicity of amino acid residues, which are nearest to the hydrolyzed peptide bond (P2 site). The improvement of prediction of hydrolysis rates performed by the used program was achieved after considering the presence of hydrophobic neighborhood of Lys48--Ile49 and Arg202--Gly203 bonds.

Quantitative comparison of casein and rapeseed proteolysis by pancreatin.

The problem of quantitative comparison of kinetic curves was solved for casein and rapeseed pancreatin hydrolysis in a membrane reactor, which ensured the measurement of proteolysis kinetics for the products with a molecular weight of less than 1000. Coordinates were derived which provided good linearization of kinetic curves and the determination of relative rate constants irrespective of reagent concentrations, E0/S0 ratio and time intervals of kinetic measurements. When the relative rate constants of the release of the individual amino acid residues in the low-weight proteolysis products were compared, trypsin-dependent constants (for Lys and Arg residues) were found to be two times less for rapeseed than for casein, and chymotrypsin-dependent constants (for Tyr and Phe residues) were approximately 1.3 times higher for rapeseed than for casein. Statistical analysis demonstrated that the distribution of constants was narrower for rapeseed than for casein. Differences between target (Arg, Lys, Tyr and Phe) and non-target constants of release in the form of peptides and free amino acids, or in the form of free amino acids only, were attributed on the differences in the peptide bond masking for casein and rapeseed proteins. Computer simulation of proteolysis kinetics was performed by PROTEOLYSIS program package to confirm the dependence of rate constant distribution on the state of masking.

[Initial kinetics of milk protein hydrolysis by chymotrypsin]. / Issledovanie nachal'noi kinetiki gidroliza belkov moloka khimotripsinom.

The kinetics of the initial stages of hydrolysis of alpha- and beta-caseins, alpha-lactalbumin, beta-lactoglobulin, and bovine serum albumin was studied quantitatively by electrophoresis. The hydrolysis rates of caseins (demasked polypeptide chains) exceeded the rates of the degradation of milk whey proteins limited by the protein globule demasking by more than two orders of magnitude. Even relatively accessible for hydrolysis globular proteins (beta-lactoglobulin and BSA) were hydrolyzed by the one-by-one mechanism. The maximum value of the ratio between the rates of the peptide chain demasking and the protein globule hydrolysis was 0.0035.

Kinetic description of proteolysis. Part 3. Total kinetics of peptide bonds hydrolysis in peptide mixtures.

A theoretical analysis of the general kinetics of peptide bond hydrolysis in peptide mixtures has been carried out. It is shown that the equation for the hydrolysis rate must include ensemble-averaged values which change as functions during hydrolysis. Thus the problem of describing the hydrolysis is reduced to finding these functions. As an example of theoretical prediction for the behavior of the averaged functions the procedure of calculating the averaged constant of chymotrypsin substrate specificity as a function of the degree of hydrolysis is presented. The analysis performed enables to find factors responsible for the decrease of hydrolysis rate, in particular, the role of the substrate specificity range and the S/E ratio. The design of specific experiments making it possible to measure the averaged values in the course of hydrolysis has been considered.

Kinetic description of proteolysis. Part 4. Hydrolysis kinetics of partial protein hydrolysates.

This report presents experimental data on kinetics of casein hydrolysate hydrolysis. The experiments were interpreted in the framework of a theory reported, which is guided by an experimentally measurable value--a total molar concentration of all peptides and amino acids (total amino nitrogen). The total amino nitrogen N was determined by photometry of N-trinitrophenyl (N-TNP) derivatives obtained by means of trinitrobenzenesulfonic acid (TNBS). Unlike the procedures reported previously, we stopped the proteolysis reaction by boiling, and performed the trinitrophenylation in a 20% V. aqueous solution of MeCN.

Kinetic description of proteolysis. Part 1. Peptic hydrolysis of proteins isolated from chicken heart: optimization in terms of time and substrate concentration.

Simple equation for the total rate of proteolysis is proposed. The equation, based on the renal mechanism of proteolysis includes the substrate concentration and some functions of the degree of hydrolysis. These functions are experimentally determined in the case of peptic hydrolysis of chicken heart proteins. The integration of differential equation made it possible to describe the kinetics of the proteolysis in terms of substrate concentration, degree of proteolysis and reaction time. The kinetics of proteolysis does not obey the Michaelis-Menten law. The proposed way for mathematical modelling permits the optimization by productivity.

Kinetic description of proteolysis. Part 2. Substrate regulation of peptide bond demasking and hydrolysis. Liquid chromatography of hydrolyzates.

Lower peptides and amino acids in hydrolyzates of casein obtained with protosubtilin were determined using high pressure liquid chromatography (HPLC). The dependences of product concentrations in hydrolyzates produced at different substrate concentrations on the degree of hydrolysis were obtained. With an increase of the substrate concentration the real type of proteolysis changes from "zipper" to "one-by-one". This result supports the assumption that the substrate proteolysis regulation is realized through the change in the ratio of the rates of peptide bond demasking to those of peptide bond hydrolysis.

[Mathematical model of protoplasm flow in a viscous-elastic active strand of a myxomycete plasmodium]. / Matematicheskaia model' techeniia protoplazmy v viazkouprugom aktivnom tiazhe plazmodiia miksomitseta.

A mathematical model is plotted of protoplasmic flow in long strands of Myxomycete plasmodium. An analytical relationship was obtained between the characteristics of protoplasmic flow: amplitude and contour of velocity in the strand channel with the amplitude and wave length of pressure developed by contractile filaments of the strand cortical layer. The model permitted a comparison to be made between the experimental data of protoplasmic flow obtained by optical methods and the evidence on contractile apparatus obtained by tensiometric measurements. A conclusion is drawn on the consistency of the basic hypothesis concerning an autowave pattern of the motive force of the cortical layer filaments.