APPLICATION OF THE METHOD OF THERMAL-DENATURATION FOR INVESTIGATION OF ALPHA-CHYMOTRYPSIN ADDUCTS WITH POLY(ALKYLENE OXIDES)

The thermostability of conjugates, non-covalent complexes and mixtures of alpha-chymotrypsin (alpha-ChT) with poly(alkylene oxides)-poly(eth ylene glycol) (PEG) with molecular mass of 1.9 kD and diblock copolyme rs of ethylene and propylene oxides (proxanols)-has been investigated. It was shown that the addition of PEG in concentration up to 2 wt. % to the solution of alpha-ChT did not affect the rate of the enzyme the rmoinactivation. Meanwhile the addition of proxanol in the same concen tration resulted in twofold decrease in the rate constant for the slow inactivation step, k(2). Even more pronounced decrease in the thermoi nactivation rate was observed for alpha-ChT-proxanol complexes obtaine d by heating or under the action of high pressure. The general tendenc y in the behavior of complexes of both types was the decrease in the k (2) constant as the temperature or pressure used for complex preparati on increased. The highest stabilizing effect was observed for complex obtained by heating up to 52 degrees C and containing maximal number o f polymer chains (molar ratio proxanol/alpha-ChT was 10). For this com plex fourfold decrease in the k(2) value was observed. Covalent attach ment of PEG or proxanol to enzyme gives maximal stabilizing effect wit h up to tenfold decrease in the k(2) value. The investigation of the t hermal denaturation kinetics of alpha-ChT and its adducts with poly(al kylene oxides) by means of fluorescence spectroscopy has shown that th e presence of polymer chains practically does not affect the rate of p rotein denaturation registered by the decrease in the intensity of pro tein fluorescence. The polymer chains, probably, diminish the rate of melting of the active site-containing region of the protein molecule. At the same time, the overall denaturation rate is independent of the presence of polymer chains in the vicinity of the protein globule.