Correlation of high‐temperature stability of α‐chymotrypsin with ‘salting‐in’ properties of solution

Abstract

A correlation between the stability of a-chymotrypsin against irreversible thermal inactivation at high temperatures (long-term stability) and the coefficient of Setchenov equation as a measure of salting-idout efficiency of solutes in the Hofmeister series has been found. An increase in the concentration of salting-in solutes (KSCN, urea, guanidinium chloride, formamide) leads to a manyfold decrease of the inactivation rate of the enzyme. In contrast, addition of salting-out solutes has a small effect on the long-term stability of a-chymotrypsin at high temperatures. The effects of solutes are additive with respect to their salting-idout capacities ; the stabilizing action of the solutes is determined by the calculated Setchenov coefficient of solution. The correlation is explained by a solute-driven shift of the conformational equilibrium between the 'low-temperature' native and the 'high-temperature' denatured forms of the enzyme within the range of the kinetic scheme put forward in the preceding paper in this journal : irreversible inactivation of the high-temperature form proceeds much more slowly compared with the low-temperature form.a-Chymotrypsin is one of the favorite subjects for studies of the mechanisms of protein stability [l-41 and stabilization [5] (and references therein). Nevertheless, the influence of low-molecular-mass compounds on irreversible thermoinactivation of this enzyme (long-term stability) has not been studied at high temperatures (60-80°C). Such studies may provide a better practical application of enzymes because it is often necessary to preserve an active enzyme from inactivation during treatment at high temperatures. The preparation of protease-containing detergents, sterilization of medicines and some other biotechnological processes which include prolonged heating can be mentioned as examples [6]. On the other hand, even at lower temperatures (4O-5O0C), the effects on proteins of urea and guanidinium chloride (GdmC1) [7-91, salts [lo-121, organic solvents [13-151 and other solutes are very different and cannot be described easily by a general theory (for one of the best recent attempts, see [16]). Thus, a better understanding of denaturation at high temperatures may be welcomed from a theoretical point of view [17].The principal possibility of regulating long-term stability by solutes may be based on the idea that enzyme inactivation is a multi-step process often involving the reversible unfolding of the protein molecule [18-201. If in the folded native state and in the unfolded reversibly denatured state an enzyme is inactivated at different rates, one may try to control inactivation by shifting the conformational equilibrium between these states. The equilibrium constant that governs the reversible unfolding of proteins is influenced significantly by low-molecular-mass additives [14, 211. An ability of ions to favor either the folded or the unfolded state is determined qualitatively by their position in the Hofmeister series, on condition that ions do not preferentially ...

Multiple amino acid substitutions significantly improve the thermostability of feruloyl esterase A from Aspergillus niger

“…A similar unusual thermal inactivation pattern has been reported for a-chymotrypsin as the ''zig-zag'' temperature dependence of the rate constant of irreversible thermoinactivation (Levitsky et al, 1994a;Levitsky et al, 1994b). A possible explanation for this phenomenon is that the thermal inactivation of the enzyme at low temperature and high temperature are subjected to different mechanisms and the 'high-temperature' denatured form is more stable against irreversible thermoinactivation than the ''low-temperature'' form (Levitsky et al, 1994a).…”

Buffers more than buffering agent: introducing a new class of stabilizers for the protein BSA

“…85 The molecular weight of CT is 25 kDa. The The value of the activity was obtained from at least three repeated measurements for each sample at a given temperature.…”

Thermal stability of immobilized ?-chymotrypsin is additionally increased by chaotropic compounds

“…'Mildly or moderately' bound enzymes (by 2 or 6 bonds, respectively) are stabilized by KSCN or GdmCl (points 1-3 in Fig. 3), as expected from the analogy with free achymotrypsin (Levitsky et al, 1994b). The level of stability thus attained is somewhat higher than that achieved by using sole immobilization.…”

“…As shown with water-soluble derivatives of a-chymotrypsin (Mozhaev et al, 1992;Levitsky et al, 1994b), conformational equilibrium between forms N and D, and consequently stability of the enzyme to irreversible thermal inactivation, can be changed by addition of low-molecularweight compounds. Chaotropic salts proved to be very effective in displacing this conformational equilibrium towards the stable form D. The effect of two typical chaotropic salts, potassium thiocyanate (KSCN) and guanidinium chloride (GdmCl), on thermal stability of immobilized a-chymotrypsins is shown in Fig.…”