Weiss R H, Flickinger A G, Rivers W J, Hardy M M, Aston K W, Ryan U S, Riley D P
Department of Chemical Sciences, Monsanto Company, St. Louis, Missouri 63167.
J Biol Chem. 1993 Nov 5;268(31):23049-54.
By stopped-flow kinetic analysis, we have directly evaluated the superoxide dismutase (SOD) activity of a number of organic nitroxides and iron- and manganese-based complexes that have been attributed with having SOD activity based upon competition experiments with cytochrome c. In 60 mM HEPES buffer, pH 8.1, or 50 mM potassium phosphate buffer, pH 7.8, Mn(II) and manganese complexes of desferal had no detectable SOD activity by stopped-flow analysis (catalytic rate constant (kcat) < 10(5.5) M-1 s-1), whereas Mn(II) and manganese complexes of desferal inhibited the reduction of cytochrome c by superoxide generated by the xanthine/xanthine oxidase system. Fe(II)-N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (FeTPEN) was eight times more active than Fe(III)-tris[N-(2-pyridylmethyl)-2-aminoethyl]amine(Fe-TPAA) in the cytochrome c assay, but only FeTPAA catalyzed the first-order decay of superoxide (kcat = 2.15 x 10(6) M-1 s-1) by stopped-flow. Fe(III)-tetrakis(4-N-methylpyridyl)porphine (FeTMPP) was active at low micromolar concentrations in both the cytochrome c and stopped-flow assays. At high micromolar concentrations, the organic nitroxides 2,2,6,6-tetramethylpiperidin-1-yloxy (TEMPO) and 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-yloxy (TEMPOL) were inhibitory in the cytochrome c assay, but showed no detectable SOD activity by stopped-flow. None of the tested compounds inhibited xanthine oxidase activity as shown by the measurement of urate production. Under the conditions of the cytochrome c assay, FeTPEN, TEMPO, and TEMPOL oxidized reduced cytochrome c which rationalizes the false positives for these compounds in this assay. The inhibitory activities of Mn(II) and the manganese desferal complexes in the cytochrome c assay appear to be due to a stoichiometric, not catalytic, reaction with superoxide as catalytic amounts of these agents do not induce a first-order decay of superoxide as shown by stopped-flow.
通过停流动力学分析，我们直接评估了许多有机氮氧化物以及铁基和锰基配合物的超氧化物歧化酶（SOD）活性，这些物质基于与细胞色素c的竞争实验而被认为具有SOD活性。在pH 8.1的60 mM HEPES缓冲液或pH 7.8的50 mM磷酸钾缓冲液中，去铁胺的Mn（II）和锰配合物通过停流分析未检测到SOD活性（催化速率常数（kcat）<10(5.5) M-1 s-1），而去铁胺的Mn（II）和锰配合物抑制了黄嘌呤/黄嘌呤氧化酶系统产生的超氧化物对细胞色素c的还原。在细胞色素c测定中，Fe（II）-N，N，N'，N'-四（2-吡啶甲基）乙二胺（FeTPEN）的活性比Fe（III）-三[N-（2-吡啶甲基）-2-氨基乙基]胺（Fe-TPAA）高八倍，但通过停流法只有FeTPAA催化超氧化物的一级衰变（kcat = 2.15 x 10(6) M-1 s-1）。Fe（III）-四（4-N-甲基吡啶基）卟啉（FeTMPP）在细胞色素c测定和停流测定中在低微摩尔浓度下都具有活性。在高微摩尔浓度下，有机氮氧化物2,2,6,6-四甲基哌啶-1-氧基（TEMPO）和4-羟基-2,2,6,6-四甲基哌啶-1-氧基（TEMPOL）在细胞色素c测定中具有抑制作用，但通过停流法未显示可检测到的SOD活性。如尿酸生成的测量所示，所测试的化合物均未抑制黄嘌呤氧化酶活性。在细胞色素c测定的条件下，FeTPEN、TEMPO和TEMPOL氧化还原型细胞色素c，这解释了这些化合物在该测定中的假阳性结果。细胞色素c测定中Mn（II）和去铁胺锰配合物的抑制活性似乎是由于与超氧化物的化学计量反应，而非催化反应，因为这些试剂的催化量并未如停流法所示诱导超氧化物的一级衰变。
