Pedrajas J R, Peinado J, López-Barea J
Departamento de Bioquímica y Biología Molecular Facultad de Veterinaria, Universidad de Córdoba, Spain.
Free Radic Res Commun. 1993;19(1):29-41. doi: 10.3109/10715769309056496.
Liver cell-free extracts of fish (Mugil sp.) from polluted environments show new Cu,Zn-SOD isoenzymes when analyzed by polyacrylamide gel electrophoresis or isoelectrofocusing followed by in situ staining for SOD activity. The most active isoenzymes, with pI 6.1 and 5.1, were present both in control and problem samples while the isoenzymes of intermediate pI value showed significant differences. Fish from control areas 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 their utility as biomarkers, Cu,Zn-SOD isoenzymes from polluted fish livers were purified to homogeneity. Five superoxide dismutase peaks were purified, named thereafter I (pI 6.1) to V (pI 5.1) respectively. Isoenzymes I and V displayed the highest specific activity. Upon incubation with moderate H2O2 concentrations, pure isoenzyme I yielded more acidic bands with pI 5.5, 5.45 and 5.35, this last being predominant. The pure isoenzyme V generated only a new band of pI 5.0. Concomitant with oxidation, the activity of peaks I and V was lost in a H2O2 concentration-dependent manner. The pattern of the new acidic bands generated upon the oxidixing treatment of isoenzyme I closely resembles that observed in crude extracts from polluted animals.
对来自污染环境的鲻鱼（鲻属）肝细胞游离提取物进行聚丙烯酰胺凝胶电泳或等电聚焦分析，随后进行超氧化物歧化酶（SOD）活性原位染色时，会显示出新的铜锌超氧化物歧化酶同工酶。最具活性的同工酶，其等电点（pI）为6.1和5.1，在对照样本和问题样本中均有出现，而中等pI值的同工酶则表现出显著差异。来自对照区域的鱼显示出三种中等pI值的同工酶，其pI分别为5.7、5.5和5.4（最后一种相当微弱），而受污染的鱼则显示出pI为5.9、5.45和5.35的三条带，最后一条非常明显。为了进一步确定它们作为生物标志物的效用，对受污染鱼肝中的铜锌超氧化物歧化酶同工酶进行了纯化，直至达到均一性。纯化出了五个超氧化物歧化酶峰，此后分别命名为I（pI 6.1）至V（pI 5.1）。同工酶I和V表现出最高的比活性。在与中等浓度的过氧化氢孵育后，纯同工酶I产生了更多pI为5.5、5.45和5.35的酸性条带，最后一条为主导。纯同工酶V仅产生了一条新的pI为5.0的条带。随着氧化过程，峰I和V的活性以过氧化氢浓度依赖的方式丧失。同工酶I经氧化处理后产生的新酸性条带模式与受污染动物粗提物中观察到的模式非常相似。
