ENZYME-POLYELECTROLYTE COMPLEXES IN WATER-ETHANOL MIXTURES - NEGATIVELY CHARGED GROUPS ARTIFICIALLY INTRODUCED INTO ALPHA-CHYMOTRYPSIN PROVIDE ADDITIONAL ACTIVATION AND STABILIZATION EFFECTS

Formation of noncovalent complexes between alpha-chymotrypsin (CT) and a polyelectrolyte, polybrene (PB), has been shown to produce two majo r effects on enzymatic reactions in binary mixtures of polar organic c osolvents with water. (i) At moderate concentrations of organic cosolv ents (10% to 30% v/v), enzymatic activity of CT is higher than in aque ous solutions, and this activation effect is more significant for CT i n complex with PB (5- to 7-fold) than for free enzyme (1.5- to 2.5-fol d). (ii) The range of cosolvent concentrations that the enzyme tolerat es without complete loss of catalytic activity is much broader. For en hancement of enzyme stability in the complex with the polycation, the number of negatively charged groups in the protein has been artificial ly increased by using chemical modification with pyromellitic and succ inic anhydrides. Additional activation effect at moderate concentratio ns of ethanol and enhanced resistance of the enzyme toward inactivatio n at high concentrations of the organic solvent have been observed for the modified preparations of CT in the complex with PB as compared wi th an analogous complex of the native enzyme. Structural changes behin d alterations in enzyme activity in water-ethanol mixtures have been s tudied by the method of circular dichroism (CD). Protein conformation of all CT preparations has not changed significantly up to 30% v/v of ethanol where activation effects in enzymatic catalysis were most pron ounced. At higher concentrations of ethanol, structural changes in the protein have been observed for different forms of CT that were well c orrelated with a decrease in enzymatic activity. (C) 1997 John Wiley & Sons, Inc.