Iqbal J, Whitney P
Calvin and Flavia Oak Asthma Research and Treatment Facility, University of Miami School of Medicine, FL 33136.
Free Radic Biol Med. 1991;10(1):69-77. doi: 10.1016/0891-5849(91)90023-v.
Eucaryotes have two major forms of superoxide dismutase (SOD), Cu,ZnSOD and MnSOD; in most tissues Cu,ZnSOD is present in higher amounts than MnSOD. To assay MnSOD, Cu,ZnSOD can be inhibited selectively by millimolar concentrations of cyanide ion. However, calculation of MnSOD activity from the differential cyanide inhibition assay is complex and small experimental errors can cause large errors in the calculated MnSOD activity. We have assessed how interaction of cyanide and hydrogen peroxide with cytochrome c can lead to further errors in the xanthine oxidase-cytochrome c assay for SOD. Alternatively, Cu,ZnSOD can be completely inactivated by 50 mM diethyldithiocarbamate (DDC) at 30 degrees C for 1 h without affecting the activity of MnSOD. Since DDC reduces cytochrome c, the treated samples must be thoroughly dialyzed or desalted before assay. In the case of lung homogenates, dialysis is not an extra step since fresh, untreated samples must also be dialyzed or desalted before assaying by the cytochrome c method. Cu,ZnSOD activity is equal to the activity in the untreated sample minus the activity in the DDC-treated portion of the sample. Another copper chelator, triethylenetetramine, did not inactivate Cu,ZnSOD and could not be used instead of DDC. For accurate measurement of both enzymes in samples where MnSOD contributes only a small fraction of the total SOD activity, the DDC method has the advantage that it provides a direct measure of the MnSOD activity without interference by Cu,ZnSOD.
真核生物有两种主要形式的超氧化物歧化酶（SOD），即铜锌超氧化物歧化酶（Cu,ZnSOD）和锰超氧化物歧化酶（MnSOD）；在大多数组织中，Cu,ZnSOD的含量高于MnSOD。为了测定MnSOD，毫摩尔浓度的氰离子可选择性抑制Cu,ZnSOD。然而，通过氰化物差异抑制测定法计算MnSOD活性很复杂，且小的实验误差会导致计算出的MnSOD活性出现较大误差。我们评估了氰化物和过氧化氢与细胞色素c的相互作用如何导致在用于SOD的黄嘌呤氧化酶 - 细胞色素c测定中产生进一步误差。另外，在30℃下，50 mM二乙基二硫代氨基甲酸盐（DDC）可使Cu,ZnSOD完全失活1小时，而不影响MnSOD的活性。由于DDC会还原细胞色素c，因此处理后的样品在测定前必须彻底透析或脱盐。对于肺匀浆，透析并非额外步骤，因为新鲜的未处理样品在通过细胞色素c方法测定之前也必须进行透析或脱盐。Cu,ZnSOD活性等于未处理样品中的活性减去样品中DDC处理部分的活性。另一种铜螯合剂三亚乙基四胺不能使Cu,ZnSOD失活，因此不能替代DDC使用。对于准确测量样品中两种酶，其中MnSOD仅占总SOD活性的一小部分，DDC方法的优点是它可直接测量MnSOD活性，而不受Cu,ZnSOD的干扰。
