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Based on the idea that proteins can be stabilized by a decrease in the thermodynamically unfavorable contact of the hydrophobic surface clusters with water, alpha-chymotrypsin (CT) was acylated with carboxylic acid anhydrides or reductively alkylated with aliphatic aldehydes. Modification of CT with hydrophilic reagents leads to 100-1000-fold increase in stability against the irreversible thermoinactivation. The correlation holds: the greater the hydrophilization increment brought about by the modification, the higher is the protein thermostability. After some limiting value, however, a further increase in hydrophilicity does not change thermostability.It follows from the dependence of the thermoinactivation rate constants on temperature that for hydrophilized CT there is the conformational transition at 55-65 degrees C into an unfolded state in which inactivation is much slower than that of the low-temperature conformation. The thermodynamic analysis and fluorescent spectral data confirm that the slow inactivation of hydrophilized CT at high temperatures proceeds via a chemical mechanism rather than Incorrect refolding operative for both the native and low-temperature form of the modified enzyme. Hence, the hydrophilization stabilizes the unfolded high-temperature conformation by eliminating the incorrect refolding.

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 ...

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We have obtained unusual 'zig-zag' temperature dependencies of the rate constant of irreversible thermoinactivation (kJ of enzymes (a-chymotrypsin, covalently modified a-chymotrypsin, and ribonuclease) in a plot of log k,, versus reciprocal temperature (Arrhenius plot). These dependencies are characterized by the presence of both ascending and descending linear portions which have positive and negative values of the effective activation energy ( E J , respectively. A kinetic scheme has been suggested that fits best for a description of these zig-zag dependencies. A key element of this scheme is the temperature-dependent reversible conformational transition of enzyme from the 'lowtemperature' native state to a 'high-temperature' denatured form; the latter form is significantly more stable against irreversible thermoinactivation than the native enzyme. A possible explanation for a difference in thermal stabilities is that low-temperature and high-temperature forms are inactivated according to different mechanisms. Existence of the suggested conformational transition was proved by the methods of fluorescence spectroscopy and differential scanning calorimetry. The values of AH and AS for this transition, determined from calorimetric experiments, are highly positive ; this fact underlies a conclusion that this heat-induced transition is caused by an unfolding of the protein molecule. Surprisingly, in the unfolded high-temperature conformation, a-chymotrypsin has a pronounced proteolytic activity, although this activity is much smaller than that of the native enzyme.It is well known that temperature dependencies of the rates of most reactions with enzyme participation, likewise usual chemical reactions, follow the Arrhenius equation [l]. Consequently, these temperature dependencies are strictly linear in a plot of log v (or log k) versus T', where v is the reaction rate, k is the corresponding rate constant, and T is absolute temperature. Deviations from the linear dependencies are not often encountered and are usually due to changes in the reaction mechanism or catalyst structure, or some other factors.Nonlinear temperature dependencies have sometimes also arisen in studies of the irreversible thermoinactivation of enzymes [2-41. Unlike reversible thermal denaturation, which usually is a single-step conformational transition between two states of a protein [5] recent reviews]. As a consequence, the apparent rate of irreversible thermoinactivation, v,,, (and the corresponding rate constant, kin) depends in a complicated manner on the rates of the processes which participate in thermal inactivation. These processes to different extents depend on temperature and this fact can explain appearance of nonlinear dependencies of v,, versus T-' [3, 41.In this study we observed temperature dependencies of the rates of irreversible thermal inactivation of a-chymotrypsin and ribonuclease of zig-zag type and have put forward a kinetic model that describes this unusual behavior. MATERIALS AND METHODS Materialsa-Chymotrypsin (Sigma,...