Unfolding and Inactivation of Abalone (Haliotis diversicolor) Alkaline Phosphatase During Denaturation by Guanidine Hydrochloride

Abstract

Abalone, a kind of low poikilothermic invertebrate, is easily exposed to ocean environment stress. Since it is one of the important mariculture animals, the attention paid to the abalone study becomes increasing. Alkaline phosphatase (ALPase, EC 3.1.3.1) is a kind of zinc-contained metalloenzyme, which catalyzes the nonspecific hydrolysis of phosphate monoesters. Unfolding and inactivation of ALPase from abalone (Haliotis diversicolor) during denaturation by guanidine hydrochloride (GuHCl) of different concentrations has first been studied. The kinetic theory of the substrate reaction by enzyme was described by Tsou, which was applied to the study on ALPase's kinetic course of inactivation by GuHCl. The result showed that the inactivation of the enzyme by GuHCl was a slow, reversible reaction with fractional remaining activity. The microscopic rate constants were determined. The result, k(+0) > k'(+0), showed that the enzyme was protected by the substrate to a certain extent during guanidine denaturation. The changes of conformation of the enzyme in different concentrations of GuHCl have been studied by means of measuring the fluorescence spectra. The results showed that the inactivation occurred before the noticeable conformational changes of the enzyme molecule as a whole can be detected, which suggests that the active site of the enzyme has more flexibility than the whole enzyme molecule. These studies will facilitate the understanding of physiological and biochemical features of the H. diversicolor and will also help in the understanding of the abalone immune system.

Cellulase Hydrolysis of Rice Straw and Inactivation of Endoglucanase in Urea Solution

“…Enzyme denaturation was carried out at 4 °C in solutions containing different concentrations of urea in 0.01 M Tris-HCl buffer (pH 7.5) for 24 h. Denatured enzyme solutions were used for activity assay and measurement of fluorescence spectra. 11 Fluorescence spectra were obtained with a Hitachi 850 spectrofluorometer. The excitation wavelength was 279 nm.…”