a-Chymotrypsin a-CT. activity was tested in aqueous media with the following cetyltrialkylammonium bromide surfactants in the series methyl, ethyl, propyl and butyl, different in the head group size, and for the sake of comparison also with the anionic sodium n-dodecyl sulfate and the zwitterionic myristyldimethylammonium propanesulfonate. N-glutaryl-Lphenylalanine p-nitroanilide hydrolysis rate was monitored at surfactant concentration above the critical micellar one. Only some cationic surfactants gave superactivity and the head group size had a major weight. The highest superactivity was measured in the presence of cetyltributylammonium bromide. The effect of both nature and concentration of three different buffers was also investigated. There is a dependence of enzyme superactivity on buffer type. Michaelis–Menten kinetics were found. The binding constants of substrate with micellar aggregates were determined in the used buffers and the effective improvement of reaction rate at the same free substrate concentration in the medium. was calculated. kcat significantly increased while Km was little changed after correction to free substrate concentration. The ratio of kcat to Km was between 12 and 35 times higher than in pure buffer, depending on buffer and surfactant concentrations. The increase of a-CT activity 30%. was less important in the presence of 1=10y2 M tetrabutylammonium bromide, a very hydrophobic salt, unable to micellise. Fluorescence spectra showed differences of enzyme conformation in the presence of various surfactants. q1999 Published by Elsevier Science B.V. All rights reserved.
alfa-Chymotripsin Superactivity in Aqueous Solutions of Cationic Surfactans
GERMANI, Raimondo;SAVELLI, Gianfranco
1999
Abstract
a-Chymotrypsin a-CT. activity was tested in aqueous media with the following cetyltrialkylammonium bromide surfactants in the series methyl, ethyl, propyl and butyl, different in the head group size, and for the sake of comparison also with the anionic sodium n-dodecyl sulfate and the zwitterionic myristyldimethylammonium propanesulfonate. N-glutaryl-Lphenylalanine p-nitroanilide hydrolysis rate was monitored at surfactant concentration above the critical micellar one. Only some cationic surfactants gave superactivity and the head group size had a major weight. The highest superactivity was measured in the presence of cetyltributylammonium bromide. The effect of both nature and concentration of three different buffers was also investigated. There is a dependence of enzyme superactivity on buffer type. Michaelis–Menten kinetics were found. The binding constants of substrate with micellar aggregates were determined in the used buffers and the effective improvement of reaction rate at the same free substrate concentration in the medium. was calculated. kcat significantly increased while Km was little changed after correction to free substrate concentration. The ratio of kcat to Km was between 12 and 35 times higher than in pure buffer, depending on buffer and surfactant concentrations. The increase of a-CT activity 30%. was less important in the presence of 1=10y2 M tetrabutylammonium bromide, a very hydrophobic salt, unable to micellise. Fluorescence spectra showed differences of enzyme conformation in the presence of various surfactants. q1999 Published by Elsevier Science B.V. All rights reserved.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
