Baez S, Linderson Y, Segura-Aguilar J
Department of Biochemistry, Wallenberg Laboratory, Stockholm University, Sweden.
Chem Biol Interact. 1994 Nov;93(2):103-16. doi: 10.1016/0009-2797(94)90090-6.
Dopa was oxidized by Mn(3+)-pyrophosphate complex to the corresponding o-quinone, accompanied by the cyclization of the amino chain to form cyclized dopa ortho-quinone (cDoQ) with absorption maxima at wavelengths of 305 and 475 nm. The cyclization was found to proceed in a single step from DoQ to cDoQ without formation of cDoQH2 and oxygen consumption. DT-diaphorase catalyzes the reduction of cDoQ to the corresponding hydroquinone (cDoQH2), which was found to be unstable in the presence of oxygen. The autoxidation of the cDoQH2 was followed by recording the constant oxidation of NADH and oxygen consumption and reduction of cDoQ at a wavelength of 475 nm. It was found that three different oxidizing agents were involved in autoxidation of cDoQH2. The addition of DETAPAC resulted in a strong inhibition of NADH oxidation (65% inhibition) during the reduction of cDoQ by DT-diaphorase, suggesting that manganese was responsible for 65% of the autoxidation of cDoQH2. The addition of SOD to the incubation mixture resulted in the inhibition of NADH oxidation (79%) during the reduction of cDoQ by DT-diaphorase. In the presence of DETAPAC, the addition of SOD inhibited NADH oxidation during cDoQH2 autoxidation 75%, suggesting that superoxide radicals are responsible for 75% of the oxygen-dependent autoxidation. The remaining NADH oxidation, which was not inhibited by DETAPAC and SOD, was accompanied by a constant oxygen consumption, suggesting that this autoxidation of cDoQH2 proceeds by reducing oxygen to superoxide radical. The effect of SOD and catalase in the presence of DETAPAC was also studied. A nearly complete inhibition (90%) of oxygen consumption during the reduction of cDoQ by DT-diaphorase was observed when SOD alone or SOD and catalase were added to the incubation mixture containing DETAPAC. We conclude that SOD and catalase constitute a protective cellular system against formation of reactive oxygen species during reduction of cDoQ by DT-diaphorase.
多巴被焦磷酸锰（Ⅲ）络合物氧化为相应的邻醌，同时氨基链环化形成环化多巴邻醌（cDoQ），其在305和475 nm波长处有吸收最大值。发现环化反应从多巴醌（DoQ）到cDoQ一步完成，不形成cDoQH2且不消耗氧气。DT - 黄递酶催化cDoQ还原为相应的对苯二酚（cDoQH2），发现其在有氧存在时不稳定。通过记录NADH的持续氧化、氧气消耗以及在475 nm波长处cDoQ的还原情况来跟踪cDoQH2的自氧化过程。发现cDoQH2自氧化过程中有三种不同的氧化剂参与。添加DETAPAC导致在DT - 黄递酶还原cDoQ过程中NADH氧化受到强烈抑制（65%抑制），表明锰导致了cDoQH2 65%的自氧化。向孵育混合物中添加超氧化物歧化酶（SOD）导致在DT - 黄递酶还原cDoQ过程中NADH氧化受到抑制（79%）。在存在DETAPAC的情况下，添加SOD抑制了cDoQH2自氧化过程中75%的NADH氧化，表明超氧自由基导致了75%的氧依赖性自氧化。未被DETAPAC和SOD抑制的其余NADH氧化伴随着持续的氧气消耗，表明cDoQH2的这种自氧化是通过将氧气还原为超氧自由基进行的。还研究了在存在DETAPAC的情况下SOD和过氧化氢酶的作用。当单独添加SOD或同时添加SOD和过氧化氢酶到含有DETAPAC的孵育混合物中时，观察到在DT - 黄递酶还原cDoQ过程中氧气消耗几乎完全受到抑制（90%）。我们得出结论，SOD和过氧化氢酶构成了一个细胞保护系统，可防止在DT - 黄递酶还原cDoQ过程中活性氧的形成。
