PURIFICATION OF CU, ZN-SUPEROXIDE DISMUTASE ISOENZYMES FROM FISH LIVER - APPEARANCE OF NEW ISOFORMS AS A CONSEQUENCE OF POLLUTION

Liver cell-free extracts of fish (Mugil sp.) from polluted environment s show new Cu,Zn-SOD isoenzymes when analyzed by polyacrylamide gel el ectrophoresis or isoelectro focusing followed by in situ staining for SOD activity. The most active isoenzymes, with pI 6.1 and 5.1, were pr esent both in control and problem samples while the isoenzymes of inte rmediate pI value showed significant differences. Fish from control ar eas showed three intermediate isoenzymes with pI 5.7, 5.5 and 5.4 (the last one quite faint) while polluted animals showed three bands of pI 5.9, 5.45 and 5.35, this last very intense. To further characterize t heir utility as biomarkers, Cu,Zn-SOD isoenzymes from polluted fish li vers were purified to homogeneity. Five superoxide dismutase peaks wer e purified, named thereafter I (pI 6.1) to V (pI 5.1) respectively. Is oenzymes I and V displayed the highest specific activity. Upon incubat ion with moderate H2O2 concentrations, pure isoenzyme I yielded more a cidic bands with pI 5.5, 5.45 and 5.35, this last being predominant. T he pure isoenzyme V generated only a new band of pI 5.0. Concomitant w ith oxidation, the activity of peaks I and V was lost in a H2O2 concen tration-dependent manner. The pattern of the new acidic bands generate d upon the oxidixing treatment of isoenzyme I closely resembles that o bserved in crude extracts from polluted animals.