Zang L Y, Misra H P
Department of Biomedical Sciences, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg 24061-0442.
J Biol Chem. 1992 Nov 25;267(33):23601-8.
1-Methyl-4-phenyl-2,3-dihydropyridinium (MPDP+), a metabolic product of the nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), has been shown to generate superoxide radicals during its autoxidation process. The generation of superoxide radicals was detected as a 5,5-dimethyl-1-pyrroline-N-oxide (DMPO).O2- spin adduct by spin trapping in combination with EPR techniques. The rate of formation of spin adduct was dependent not only on the concentrations of MPDP+ and oxygen but also on the pH of the system. Superoxide dismutase inhibited the spin adduct formation in a dose-dependent manner. The ability of DMPO to trap superoxide radicals, generated during the autoxidation of MPDP+, and of superoxide dismutase to effectively compete with this reaction for the available O2-, has been used as a convenient competition reaction to quantitatively determine various kinetic parameters. Thus, using this technique the rate constant for scavenging of superoxide radical by superoxide dismutase was found to be 7.56 x 10(9) M-1 s-1. The maximum rate of superoxide generation at a fixed spin trap concentration using different amounts of MPDP+ was found to be 4.48 x 10(-10) M s-1. The rate constant (K1) for MPDP+ making superoxide radical was found to be 3.97 x 10(-6) s-1. The secondary order rate constant (KDMPO) for DMPO-trapping superoxide radicals was found to be 10.2 M-1 s-1. The lifetime of superoxide radical at pH 10.0 was calculated to be 1.25 s. These values are in close agreement to the published values obtained using different experimental techniques. These results indicate that superoxide radicals are produced during spontaneous oxidation of MPDP+ and that EPR spin trapping can be used to determine the rate constants and lifetime of free radicals generated in aqueous solutions. It appears likely that the nigrostriatal toxicity of MPTP/MPDP+ leading to Parkinson's disease may largely be due to the reactivity of these radicals.
1-甲基-4-苯基-2,3-二氢吡啶鎓（MPDP+）是黑质纹状体毒素1-甲基-4-苯基-1,2,3,6-四氢吡啶（MPTP）的代谢产物，已证实在其自氧化过程中会产生超氧自由基。超氧自由基的产生通过自旋捕集结合电子顺磁共振（EPR）技术检测为5,5-二甲基-1-吡咯啉-N-氧化物（DMPO）·O₂⁻自旋加合物。自旋加合物的形成速率不仅取决于MPDP+和氧气的浓度，还取决于体系的pH值。超氧化物歧化酶以剂量依赖的方式抑制自旋加合物的形成。DMPO捕获MPDP+自氧化过程中产生的超氧自由基的能力，以及超氧化物歧化酶与该反应有效竞争可用O₂⁻的能力，已被用作一种方便的竞争反应来定量确定各种动力学参数。因此，使用该技术发现超氧化物歧化酶清除超氧自由基的速率常数为7.56×10⁹ M⁻¹ s⁻¹。使用不同量的MPDP+在固定自旋捕集剂浓度下超氧产生的最大速率为4.48×10⁻¹⁰ M s⁻¹。发现MPDP+产生超氧自由基的速率常数（K1）为3.97×10⁻⁶ s⁻¹。DMPO捕获超氧自由基的二级反应速率常数（KDMPO）为10.2 M⁻¹ s⁻¹。计算出pH 10.0时超氧自由基的寿命为1.25 s。这些值与使用不同实验技术获得的已发表值非常一致。这些结果表明，超氧自由基在MPDP+的自发氧化过程中产生，并且EPR自旋捕集可用于确定水溶液中产生的自由基的速率常数和寿命。导致帕金森病的MPTP/MPDP+的黑质纹状体毒性似乎很大程度上可能归因于这些自由基的反应性。
