alpha-chymotrypsin and dodecyltrimethylammonium

The rate of hydrolysis of N-glutaryl-L-phenylalanine p-nitroanilide (GPNA) catalyzed by alpha-chymotrypsin (alpha-CT) has been measured in aqueous solutions of dodecyltrimethylammonium bromide (DTAB) at concentrations below and above the critical micelle concentration, as well as in the absence of surfactant. Under all the conditions employed, the reaction follows a Michaelis-Menten mechanism. The presence of the surfactant leads to superactivity below and above the critical micelle concentration (CMC), with a maximum reaction rate taking place near the CMC when the results are treated in terms of the analytical concentration of the substrate. A similar behavior was observed by working with the enzyme partially deactivated in the presence of 4 M urea. After correction to take into account the partitioning of the substrate between the micelles and the external media, the activity of the enzyme tends to remain almost constant above the corresponding CMCs. This results from a compensation of a decrease in the catalytic constant (k(cat)) and a decrease in the Michaelis constant (K(M)). The behavior of alpha-CT in the hydrolysis of GPNA in DTAB solutions is at variance with that previously reported for the hydrolysis of 2-naphthyl acetate in solutions of the same surfactant (E. Abuin, E. Lissi, R. Duarte, Langmuir 19 (2003) 5374). An explanation of the different effects of the surfactant on the behavior of the enzyme with both substrates is advanced, taking into account the complexity of the mechanism of the alpha-CT-mediated reaction, more specifically, in terms of different rate-limiting steps for the formation of the measured products.

Topics: Catalysis; Chymotrypsin; Dipeptides; Hydrolysis; Kinetics; Quaternary Ammonium Compounds; Solutions; Water

In this new photometric assay fecal samples are pretreated with detergent and high concentrations of salts. The subsequent kinetic enzyme determination step involves the chromogenic substrate succinyl-Ala-Ala-Pro-Phe-4-nitroanilide. The sample pretreatment assures a nearly complete solubilization of the formerly particle-bound enzyme, thus permitting determination of the enzyme activity in either the suspension or the supernate after centrifugation. Furthermore this pretreatment enhances the enzyme activity and decreases the Km value. Results by this assay correlate well with those by classical titrimetry and the method is easily adapted to automated systems.

Topics: Autoanalysis; Buffers; Calcium Chloride; Chromogenic Compounds; Chronic Disease; Chymotrypsin; Feces; Humans; Hydrogen-Ion Concentration; Oligopeptides; Pancreatitis; Quaternary Ammonium Compounds; Reference Values; Sodium Chloride; Solubility; Spectrophotometry