Jewett S L, Rocklin A M
Department of Chemistry, California State University, Northridge 91330.
Anal Biochem. 1994 Mar;217(2):236-40. doi: 10.1006/abio.1994.1114.
N,N'-Diethyldithiocarbamate reacts with copper in the copper-zinc superoxide dismutase (EC 1.15.1.1) in polyacrylamide gels to form stable yellow-brown bands that are quantifiable at 448 nm. This method of examining superoxide dismutase has been applied to crude extracts of the enzyme obtained from red cell lysates from which hemoglobin has been removed by chloroform-ethanol precipitation. This treatment did not affect the activity and heterogeneity of purified dismutase added to lysates and recovered by the same method. The bands that develop in the dithiocarbamate-stained gels of the extracts correspond exactly to the bands of dismutase activity obtained with a positive activity stain using dianisidine, indicating that the dismutase is the only copper protein that gives rise to these bands. The amount of superoxide dismutase in the bands, determined by comparing the areas under unknown peaks to areas obtained with a standard dismutase sample, agrees with the amount predicted from indirect superoxide dismutase activity measurements. Any color in gels due to trace hemoglobin or hemoglobin degradation products is bleached overnight during staining with the dithiocarbamate.
N,N'-二乙基二硫代氨基甲酸盐与聚丙烯酰胺凝胶中铜锌超氧化物歧化酶（EC 1.15.1.1）中的铜发生反应，形成稳定的黄棕色条带，这些条带在448nm处可进行定量分析。这种检测超氧化物歧化酶的方法已应用于从红细胞裂解物中获得的粗酶提取物，其中血红蛋白已通过氯仿-乙醇沉淀法去除。这种处理不会影响添加到裂解物中并通过相同方法回收的纯化歧化酶的活性和异质性。提取物经二硫代氨基甲酸盐染色的凝胶中出现的条带与使用联茴香胺进行阳性活性染色获得的歧化酶活性条带完全对应，这表明歧化酶是产生这些条带的唯一铜蛋白。通过将未知峰下的面积与标准歧化酶样品获得的面积进行比较来确定条带中超氧化物歧化酶的量，这与从间接超氧化物歧化酶活性测量预测的量一致。在用二硫代氨基甲酸盐染色期间，凝胶中由于微量血红蛋白或血红蛋白降解产物产生的任何颜色会在一夜之间被漂白。
