Study On The Response Of Antioxidant Enzymes In Zebra Fish To Water Polluted By Heavy Metals And Organic Pollutants

Water is the indispensable componts in human life. Meanwhile water bodies receive numerous contaminants from both natural and anthropogenic sources, so that the safety of water affected human survival directly. Biomarkers are used as early warning indicators for pollutant exposure and toxic effects to establish a simple, rapid and accurate warning system for water pollution. Zebra fish, a model animal to understand toxicity of pollutants in water, was cultured in differently polluted water, and the dose-response relationship between pollutants and activity of antioxidant enzymes were studied, subsequently the separation and purification of relative enzyme were carried out in this paper.On the one hand, we measured two antioxidant enzymes such as catalase (CAT), and superoxide dismutase (SOD) of zebra fish exposed to sublethal concentrations of metals (Cu, Cd, Zn and Pb)and organic(phenol and PBA), where a lower (1/20median lethal concentration), moderate(1/10median lethal concentration) and higher(1/5median lethal concentration) concentrations were applied. On the other hand, an idiosyncratic proteinase was purified from zebra fish. The purification procedure consisted of ammonium sulfate fractionation and chromatographies induding DEAE-Sephacel, Hydroxyapatite, and Phenarose Sepharose6-Fast Flow. The results showed that at lower or moderate concentration of Cu2+, the expression level of SOD and CAT was obviously up-regulated. As time progressed, the expression of CAT decreased significantly at14days in the higher concentration group. The expression level of SOD was obviously dowm-regulated under the Cd2+stress. While At lower or moderate concentration, the expression level of CAT was obviously at14days in the higher concentration group. The expression level of SOD was obviously up-regulated at the lower or protocol concentration of Zn2+and the expression level of CAT was slightly up-regulated at7days. After that, the expression level was sharply recovered and reached the original level at14days. At the protocol concentration of Cd2+, the expression level of SOD was obviously up-regulated. The expression level of CAT was slightly up-regulated at7days. After that, the expression level was sharply declined and under the original level at14days. At the protocol concentration of phenol, the expression level of SOD and CAT was obviously up-regulated. At the protocol concentration of PBA, the expression level of CAT was obviously up-regulated, while the expression level of SOD was slightly down-regulated at7days. After that, the expression level was sharply recovered and reached the original level14days. Obtained data in the present study showed that the dose-response relationship between activities of SOD and CAT of zebra fish and Pollutants. SOD and CAT are two kinds of biomarkers which are used in monitoring heavy metal and organic toxicity.An aminopeptidase from zebra fish (Danio rerio) was purified1247-fold to homogeneity with35.4%recovery. The molecular mass of the enzyme was estimated at98kDa. Optimum temperature and pH of the enzyme were45℃and7.5, respectively. The enzyme preferentially hydrolyzed substrate Leu-MCA with Kcat/Km=4.2×106M-1S-1and an activation energy of80.5KJ·M-1, respectively. It was specifically inhibited by bestatin, puromycin and metal-chelating agents. According to the peptide mass fingerprint analysis, the enzyme was highly matched with the predicted D. rerio aminopeptidase puromycin sensitive. Combining the enzymatic characteristics, it can be presumed that the aminopeptidase of zebra fish as an intracellular biomarker which expression is affected by environ-mental stress.