Catalytic activity and denaturation of enzymes in water/organic cosolvent mixtures

Abstract

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

Denaturation capacity: a new quantitative criterion for selection of organic solvents as reaction media in biocatalysis

“…An important conclusion which can be drawn from the results obtained in this work and from the data published previously [9, 10,19,20,241 is that the observed threshold reversible denaturation of proteins by increasing concentrations of organic cosolvents represents a general phenomenon which operates regardless of the nature of specific solvents or proteins used.…”

Effect of different alcohols on stratum corneum kallikrein 5 and phospholipase A2 together with epidermal keratinocytes and skin irritation

“…In addition, in vitro testing was conducted to probe the potential effects of the individual alcohols at the molecular level. Several groups have shown the denaturing effects of alcohols on haemoglobin, myoglobin, cytochrome C, trypsin and a-chymotrypsin [4][5][6][7][8]. Interestingly, the denaturant potential correlated with the log octanol-water (Log P) values of the different alcohols.…”

“…The specific alcohol used in an ABHR may be n-propanol, isopropanol or ethanol depending on the country or region. The ability of alcohols to denature proteins has been demonstrated, and their potency in this respect depends on alcohol chain length and hydrocarbon content together with their octanol-water partition coefficients [4][5][6]. Enzyme activities have also been shown to be reduced in the presence of ethanol.…”

Peptide synthesis: chemical or enzymatic

“…Homogeneous systems are composed by a mixture of water and a water-miscible solvent (cosolvent) in which the enzyme is dissolved (Torres and Castro, 2004). Cosolvents are usually detrimental for enzyme activity at moderately high concentrations, because they tend to penetrate the aqueous microenvironment that surrounds the enzyme molecules, altering the pattern of interaction of the enzyme with the solvent and distorting its three-dimensional structure (Klibanov, 1986;Laane et al 1987a;Laane et al 1987b;Deetz and Rozzell, 1988;Zaks and Klibanov, 1988a;Zaks and Klibanov, 1988b;Mozhaev et al 1989;Levitsky et al 1999). However, polyols and glymes are notable exceptions among cosolvents (Castro, 2000;Illanes and Fajardo, 2001).…”