PROPERTIES OF ACID PHOSPHATASE FROM CARASSIAS AURATUS AND EFFECTS OF SEVERAL EFFECTORS ON THE ACTIVITY OF THE ENZYME

Acid phosphatase (ACP) is one kind of enzyme, which is not absolutely specific. It can catalyze the hydrolysis of phosphoric acid ester chemical compound in acid environment. ACP activities are involved in a variety of metabolic processes, such as molecule permeability, growth and cell differentiation. In ecotoxicology, this enzyme has been used as an indicator of intoxication because of its sensitivity to metallic salts. So the objective of this paper is to characterize the ACP from Carassias auratus, to analyze the in vitro effects of different metal ions on the enzyme activity and to estimate its potential use as a stress biomarker. This paper mainly deals with isolating, purifying ACP from viscera of Carassias auratus and studying its characters and preparing for its application. ACP was prepared and purified by means of the following techniques: n-butyl alcohol extraction, 0.3 and 0.8 amonanium sulfate precipitation, ion exchange chromatography on DEAE-32 column and gel filtration chromatography on Sephadex G-150. The preparation was shown to be homogenous on polyacrylamide gel electrophoresis. The specific activity of the enzyme was 195.06 unit per mg protein. Its molecular weight was determined to be about 33.3kD on SDS-PAGE. The kinetic characters of the enzyme had been studied. The enzyme showed an optimum pH of 4.8 with p-nitrophenylphosphate as substrate, however, high catalytic activity of the enzyme was within the pH range of 4.0—5.5. The optimal catalytic reaction temperature was 45℃, and the activity of the enzyme decreased quickly when the temperature was above 55℃. The Michaelis-Menten constant (Km) was 0.23mmol/L. Chemical modification was used to illuminate the essential amino acid in the catalytic activity of ACP from Carassias auratus. The results revealed that succinic anhydride, phenylmethylsulfony fluoride, dithiothreitol and N-bromosuccinimide had no effect on the activity of the enzyme, while bromoacetic acid and iodoacetic acid inhibited the enzyme. The inhibition of bromoacetic acid and iodoacetic acid on the activity of ACP from Carassias auratus indicated that there were histidine residues at the active site of the enzyme. Different metal ions had different effects on ACP from Carassias auratus. The positive monovalent metalions K+ and Na+ had no effect on the enzyme, while Ag+ inhibited the enzyme; The positive bivalent cations had different effects on the enzyme: Co2+ and Ni2+ had no effect on the enzyme; Mg2+, Ca2+, Ba2+ and Mn2+ activated the enzyme while Cu2+, Pb2+ and Cd2+ inhibited the enzyme. The aviation of Mg2+ and Ca2+ and the inhibition of Pb2+ and Cd2+ were depended on the concentration of metal ions. Fluorescence spectra of ACP which was incubated with Pb2+, Cd2+, Mg2+ and Ca2+ showed that effects of metal ions on the enzyme might be related to a conformational change on the enzyme. Thus these characters of ACP suggest that the enzyme is suitable to be used as a stress biomarker, but more knowledge of the physicochemical and kinetic characteristics of the enzyme activity of ACP from Carassias auratus is necessary before it can be employed as biochemical indicator of stress due to heavy metals.