The dependence of the catalytic activities of a-chymotrypsin and laccase on the concentration of organic cosolvents (alcohols, glycols and formamides) in mixed aqueous media has a pronounced threshold character: it does not change up to a critical concentration of the non-aqueous cosolvents added, yet further increase of the latter (by only a small percentage, by vol.) leads to an abrupt decrease in enzyme activity. Fluorescence studies indicate that the inactivation results from reversible conformational changes (denaturation) of the enzymes. There is a linear correlation between the critical concentration of residual water (at which the enzyme inactivation occurs in a threshold manner) and the hydrophobicity of the organic cosolvents added. A quantitative criterion is suggested for the selection of organic cosolvents to be used for enzymatic reactions in homogeneous water/organic solvent media.The behaviour of enzymes in mixed aqueous media has been studied extensively since the fifties (see the first review on this subject by Singer [l]). There are at least two reasons for this. First, such studies help us to clarify the contribution of different molecular forces to maintaining the native structure of the protein [l, 21 and to get a deeper insight into the structure/stability relationships in proteins in general [3]. Second, a number of enzyme-catalysed processes, such as syntheses of peptides and esters, transformation of some hormones, fats and steroids, etc. must be performed in media with a low water content. The reasons for this, e.g. the increase in solubility of poorly water-soluble natural and organic compounds and/or a thermodynamic shift of the chemical equilibrium toward the desired products (and other applied aspects), have been frequently discussed in the literature (for reviews, see [4 -81).A typical experiment designed to elucidate the effect of water-miscible organic solvents on proteins is usually performed as follows: increasing amounts of the organic cosolvents are added to an aqueous solution of the enzyme and the manner is studied in which its structure (as assayed by physical methods) and/or its catalytic activity change. The plots of protein spectral characteristics versus concentrations of an organic cosolvent are rather informative, since they have, as a rule, a pronounced threshold profile (for some examples, see [I, 9, 101). Hence, after a critical concentration of organic cosolvent (20-50% by vol. usually) has been achieved, the spectra1 characteristics of the protein change abruptly. This is strong evidence of conformational rearrangements in the structure of the protein, i.e. of its denaturation.Correspondence to V. V. Mozhaev, Chemistry Department, Abbreviation. C50r the concentration of solvent at which half Enzymes. a-Chymotrypsin

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Inactivation of alpha-chymotrypsin in aqueous solutions of alcohols and diols proceeds both reversibly and irreversibly. Reversible loss of the specific enzyme activity results from conformational changes (unfolding) of the enzyme detected by fluorescence spectroscopy. Multipoint covalent attachment to the matrix of polyacryl-amide gel by copolymerization method stabilizes alpha-chymotrypsin from denaturation by alcohols, the stabilizing effect increasing with the number of bonds between the protein and the support. Immobilization protects the enzyme also from irreversible inactivation by organic solvents resulting from bimolecular aggregation and autolysis.

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