Experimental approaches to modelling the enzymatic function of biological membranes are discussed. Emphasis is given to pseudohomogeneous systems such as proteolipid complexes and enzymes in organic solvents; the latter are solubilized with phospholipids or synthetic surfactants. Methods for producing and studying such micellar systems are considered. The key research problems of micellar enzymology are formulated and its relation to enzyme membranology is discussed. Finally, the new potentialities are noted of applied enzymology (biotechnology) offered by application of a colloidal solution of water in organic solvents as a microheterogeneous medium for enzymatic reactions.Until now the development of molecular enzymology has been mainly directed to studying free enzymes [l -41. In other words, the most valuable experiments aimed at elucidating the structure of catalytic centers and the physico-chemical mechanisms of biocatalysis were successful only with the enzymes isolated from the living cells in quite a pure form. However, such 'pure' experiments, as was noted time and again [5], naturally raise the question as to whether the enzyme properties observed in vitro can be correlated adequately with the conditions of its functioning in vivo. Such a doubt is quite equitable since it became clear [6 -91 that the subcellular structure and the compartmentalization of enzymes play the most important role in metabolism regulation.Thus, the essence of the contradiction is that in vitro studies of enzymes are usually conducted in water (buffer). However, in the living cell, enzymes mostly act on or near the 'water/ organic medium' interface. For instance, many enzymes, if not the majority, are located on the surface of biological membranes or inside them [lo-161. Other enzymes function in mobile complexes with macromolecular components of the cell, e.g. with proteins or polysaccharides [14 -161. Generally speaking, the boundary between 'bound' and 'diffusion-free' enzymes is rather conventional, and such a classification is more methodical (depending on the ease of the enzyme isolation) than functional (the enzyme localization in the cell) [16]. The problem is that many of the enzymes bind to the membrane surface loosely and the degree of their binding depends on the concentration of metabolites [14-191. The physiological state of the cell determines not only the adsorption of some enzymes but their possible translocation through membranes [20,21]. In the adsorbed state in vivo (on the enterocyte brushborder) extracellular enzymes were found as well, such as proteases of the digestive tract, namely trypsin and a-chymotrypsin [22, 231, which up to now have remained model subjects of the classical 'in-water' enzymology.Finally, a few words about the medium in which diffusionfree enzymes function in the cell (for a review see [24]). It is not very clear why macromolecules (immunoglobulins and albumin) injected into the living cell diffuse so slowly (as in a 60% sucrose solution) [25]. The comparison of the diffusion rat...

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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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To simulate in vitro the conditions under which enzymes act in vivo, enzyme molecules have been entrapped in hydrated reverse micelles of a surfactant in organic solvents. In this system the catalytic activity of one of the enzymes studied (peroxidase) became much higher than in water, and the specificity of the other enzyme (alcohol dehydrogenase) was dramatically altered.

We propose immobilizing enzymes into Nafion membranes by suspending the enzyme in a water-ethanol mixture with a high (>90%) ethanol content, followed by mixing with the dissolved polyelectrolyte, and finally allowing the enzyme-polyelectrolyte solution to dry at the target surface (electrode surface). Since Nafion solution was deposited from a solution where it is truly dissolved and without excessive dilution with water, the resulting membranes were more uniform and stable than those otherwise obtained. Enzyme suspensions in concentrated ethanol solutions were prepared without any prior modifications of the protein. The remaining activity after 30 min exposure to such solutions under optimal conditions was up to 100%. The stability of the enzymes in these suspensions was higher than that in aqueous solution. Electrochemical biosensors made accordingly showed a several times increased response compared with those of enzyme electrodes based on the traditional way of using excessive dilution of Nafion with water. The remaining activity, after the drying-washing cycle of the enzyme electrode made by enzyme immobilization from concentrated organic solvent, was at least 10 times higher than that of the traditional one.

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