The effects of surface and macromolecular interactions on the kinetics of inactivation of trypsin and α-chymotrypsin

Abstract

The autolysis of trypsin and alpha-chymotrypsin is accelerated in the presence of colloidal silica and glass surfaces. It is proposed that adsorption of the enzymes (favoured by electrostatic factors) results in a conformational change that renders the adsorbed enzyme more susceptible to proteolytic attack. Although the adsorbed enzymes are more susceptible to proteolysis, their activity towards low-molecular-weight substrates is not affected, indicating a relatively minor conformational change on adsorption. The rates of autolysis in solution (i.e. in ;inert' vessels) are second-order for both trypsin and alpha -chymotrypsin, with rate constants of 13.0mol(-1).dm(3).s(-1) for trypsin (in 50mm-NaCl at pH8.0 at 25 degrees C) and 10.2mol(-1).dm(3).s(-1) for alpha-chymotrypsin (in 0.1m-glycine at pH9.2 at 30 degrees C). In glass vessels or in the presence of small areas of silica surface (as colloidal silica particles), the autolysis of both trypsin and alpha-chymotrypsin can show first-order kinetics. Under these conditions, saturation of the surface occurs and the fast surface proteolytic reaction controls the overall kinetic order. However, when greater areas of silica surface are present, saturation of the surface does not occur, and, since for a considerable portion of the adsorption isotherm the amount adsorbed is approximately proportional to the concentration in solution, second-order kinetics are again observed. A number of negatively charged macromolecules have been shown similarly to increase the rate of autolysis of trypsin: thus this effect, observed initially with glass and silica surfaces, is of more general occurrence when these enzymes adsorb on or interact with negatively charged surfaces and macromolecules. These observations explain the confusion in the literature with regard to the kinetics of autolysis of alpha-chymotrypsin, where first-order, second-order and intermediate kinetics have been reported. A further effect of glass surfaces and negatively charged macromolecules is to shift the pH-activity curve of trypsin to higher pH values, as a consequence of the effective decrease in pH in the ;microenvironment' of the enzyme associated with the negatively charged surface or macromolecule.

Stabilizing biocatalysts

“…First-order deactivation cannot be assumed if aggregation (a higher-order process) is the method of deactivation. 56 At a constant TTN, a very active but rather unstable catalyst is equivalent to a slower catalyst with improved stability over time.…”

“…Dominance of aggregation or autohydrolysis in case of proteases leads to a concentration-dependent behavior that often can be fit via a second-order rate law. 56 If a first-or second-order plot does not fit the data, the apparent reaction order can be obtained via a Levenspiel plot of ln(dimensionless rate) vs. ln(1-degree of conversion) or ln(dX/dt) vs. ln(1 À X), the slope of which equals the apparent reaction order. 60 However, in the authors' own experience, the accessible range of biocatalyst concentration often does not lend itself to accurate Levenspiel plots.…”

Bayesian Data Integration Questions Classic Study on Protease Self-Digest Kinetics

“…In the present work, we focus on the kinetic aspects of the accelerated self-digest; therefore, we revisit a detailed experimental study by Johnson and Whateley of the kinetics of acceleration of trypsin self-digest in the presence of silica surfaces . We will refer to this publication as JW1981 in the remaining text.…”

“…5,7 In the present work, we focus on the kinetic aspects of the accelerated self-digest; therefore, we revisit a detailed experimental study by Johnson and Whateley of the kinetics of acceleration of trypsin self-digest in the presence of silica surfaces. 8 We will refer to this publication as JW1981 in the remaining text. In agreement with their earlier work, 3 JW1981 observed that the relative deactivation is identical for three different starting concentrations of trypsin if silica is present (Figure 1), i.e., the reaction is of the first-order.…”

Factors influencing antibody stability at solid–liquid interfaces in a high shear environment

“…8 In the case of a-chymotrypsin, GP-HPLC revealed significant amounts of low molecular weight species which accounted for the total loss in monomer peak area (data not shown). This fragmentation was attributed to the tendency of a-chymotrypsin to undergo autolysis upon adsorption at solid-liquid interfaces, 34 and explained why large aggregates were not detected by an increase in turbidity.…”