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The process of reversible denaturation of several proteins (a-chymotrypsin, trypsin, laccase, chymotrypsinogen, cytochrome c and myoglobin) by a broad series of organic solvents of different nature was investigated using both our own and literature data, based on the results of kinetic and spectroscopic measurements. In all systems studied, the denaturation proceeded in a threshold manner, i. e. an abrupt change in catalytic and/or spectroscopic properties of dissolved proteins was observed after a certain threshold concentration of the organic solvent had been reached. To account for the observed features of the denaturation process, a thermodynamic model of the reversible protein denaturation by organic solvents was developed, based on the widely accepted notion that an undisturbed water shell around the protein globule is a prerequisite for the retention of the native state of the protein. The quantitative treatment led to the equation relating the threshold concentration of the organic solvent with its physicochemical characteristics, such as hydrophobicity, solvating ability and molecular geometry. This equation described well the experimental data for all proteins tested. Based on the thermodynamic model of protein denaturation, a novel quantitative parameter characterizing the denaturing strength of organic solvents, called the denaturation capacity (DC), was suggested. Different organic solvents, arranged according to their DC values, form the DC scale of organic solvents which permits theoretical prediction of the threshold concentration of any organic solvent for a given protein. The validity of the DC scale for this kind of prediction was verified for all proteins tested and a large number of organic solvents. The experimental data for a few organic solvents, such as formamide and N-methylformamide, did not comply with equations describing the denaturation model. Such solvents form the group of so-called 'bad' solvents; reasons for the occurrence of 'bad' solvents are not yet clear. The DC scale was further extended to include also highly nonpolar solvents, in order to explain the wellknown ability of enzymes to retain catalytic activity and stability in biphasic systems of the type water/waterimmiscible organic solvent. It was quantitatively demonstrated that this ability is accounted for by the simple fact that nonpolar solvents are not sufficiently soluble in water to reach the inactivation threshold concentration.

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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