Liver cytochrome oxidase P4501A1 purification of Acipenser persicus and designing ELISA for measuring

Moon oxygenases related to cytochrome P450s are the molecular Biomarkers which have important role in Biotransformation of endogenous and exogenous compounds and catalazyin of many biological reactions. One of the important isoenzyme is cytochrome P4501A. This isoenzyme involved in metabolism of environment pollutnts such as PAHs. Because of its inducibility, it has a key tool for impact assesment of contaminants in aquatic environment. In this study, at first, that fractions containing Acipenser persicus and Huso huso isoenzyme were purified, and after that Antibodies against them were prepared. For isolation of isoenzyme fraction, Microsomes were prepared from fish liver using differential centrifugation at high speeds. microsomes were solubiized by cholat sodium and Emulgen. Extraction of this isoenzyme was done with the combinatuion of ionexchange chromatography and gelfiltration or chromatofocusing chromatography. Ion exchange chromatography and gel filtration were applied in DEAE sepharose fast flow and sephacryl S200 respectively and chromatofocusing was done at poly buffer 74 and 94 exchanger. The results of SDS-PAGE Showed that the molecular weight of isoenzyme was about 58±1 KDa. Furthermore the inmunoblotting results confirmed this subject. Isoenzyme activigy based on EROD (Ethoxyresorofin o-deethylase) reaction showed about 20-26 fold increase in enzyme activity of treated fish than control fish. The results of Elisa, Using monoclonal anti cod P4501A demonstrated the inducibility and highly elevated of its activity in treated sample more than the control fish. Mean while, the fish sample were showed the strong reaction to polyclonal antibody against beluga P4501A1 prepared in our Lab compared to monoclonal anti body.