Giulivi C, Forlin A, Bellin S, Cadenas E
Department of Molecular Pharmacology and Toxicology, School of Pharmacy, University of Southern California, Los Angeles 90033, USA.
Chem Biol Interact. 1998 Jan 30;108(3):137-54. doi: 10.1016/s0009-2797(97)00104-x.
The reaction between glutathione and 2,5-diaziridinyl-1,4-benzoquinones bearing halogen substituents at C3 and C6 was examined in terms of the formation of glutathionyl-quinone conjugates and semiquinones by HPLC with UV detection, mass spectroscopy and EPR. The reactivity of the halogen atoms toward sulfur substitution is the primary reaction leading to the formation of mono- and di-glutathionyl-substituted quinones. The relative formation of these conjugates depended on the GSH/quinone molar ratios. At GSH/quinone molar ratios below unity, the products observed were the reduced form of the parent quinone, a dithioether derivative and GSSG. Disulfide formation accounted for 60-68% of total GSH consumed. EPR analysis of these reaction mixtures showed a 5-line spectrum (1:2:3:2:1 relative intensities) with 2 equivalent N (aN = 1.98 G) and assigned to the semiquinone form of dichloro- diaziridinylbenzoquinone. Semiquinone quantification by double integration of the EPR signals and interpolation with an adequate standard revealed that the amount of semiquinone formed per GSH consumed was 0.98. At GSH/quinone molar ratios above unity (4, 10 and 100 molar excess of GSH) a pattern of products emerged consisting of 3,6-diglutathionyl quinones with two, one and no aziridinyl moieties, identified by mass spectral analysis. EPR studies revealed that these compounds were minor components of a composite EPR spectrum (a 3-line signal with 1:1:1 relative intensities, 1 equivalent N (aN = 1.73 G) and 1 H (aH = 1.45 G) or a 3-line signal with 1:2:1 relative intensities and 2 equivalent H (aH = 1.4 G). These minor components were assigned to the diglutathionyl conjugates bearing one- or no aziridinyl moiety, respectively. The major component in the EPR signal showed a 3-line spectrum (1:1:1 relative intensity) with 1 equivalent N (aN = 1.7 G) and a g shift of -0.96 G. This spectrum was assigned to a triglutathionyl conjugate of a monoaziridinylbenzoquinone. This major component was also observed when GSH/quinone mixtures were incubated with the two-electron transfer flavoprotein NAD(P)H:quinone oxidoreductase. The semiquinone signals were abolished by superoxide dismutase. In the presence of catalase, the contribution of these components to the overall EPR spectrum was equal. These data are discussed in terms of the one-electron transfer steps encompassed by thiol oxidation and semiquinone formation and the two-electron transfers inherent in sulfur substitution and aziridinyl group loss.
通过高效液相色谱-紫外检测、质谱和电子顺磁共振（EPR），从谷胱甘肽基-醌缀合物和半醌的形成方面，研究了谷胱甘肽与在C3和C6位带有卤素取代基的2,5-二氮丙啶基-1,4-苯醌之间的反应。卤素原子对硫取代的反应性是导致形成单谷胱甘肽基和双谷胱甘肽基取代醌的主要反应。这些缀合物的相对形成取决于谷胱甘肽/醌的摩尔比。在谷胱甘肽/醌摩尔比低于1时，观察到的产物是母体醌的还原形式、二硫醚衍生物和氧化型谷胱甘肽（GSSG）。二硫键的形成占消耗的总谷胱甘肽的60 - 68%。对这些反应混合物的EPR分析显示出一个五线谱（相对强度为1:2:3:2:1），有2个等效的氮（氮的超精细分裂常数aN = 1.98 G），并归属于二氯二氮丙啶基苯醌的半醌形式。通过对EPR信号进行双积分并用适当的标准物进行插值来定量半醌，结果表明每消耗一分子谷胱甘肽形成的半醌量为0.98。在谷胱甘肽/醌摩尔比高于1时（谷胱甘肽有4、10和100倍的摩尔过量），出现了由具有两个、一个和没有氮丙啶基部分的3,6-双谷胱甘肽基醌组成的产物模式，通过质谱分析鉴定。EPR研究表明，这些化合物是复合EPR谱的次要成分（一个三线信号，相对强度为1:1:1，1个等效的氮（aN = 1.73 G）和1个氢（aH = 1.45 G），或者一个三线信号，相对强度为1:2:1，2个等效的氢（aH = 1.4 G））。这些次要成分分别归属于带有一个或没有氮丙啶基部分的双谷胱甘肽基缀合物。EPR信号中的主要成分显示出一个三线谱（相对强度为1:1:1），有1个等效的氮（aN = 1.7 G），g位移为 - 0.96 G。该谱归属于单氮丙啶基苯醌的三谷胱甘肽基缀合物。当谷胱甘肽/醌混合物与双电子转移黄素蛋白NAD(P)H:醌氧化还原酶一起孵育时，也观察到了这个主要成分。超氧化物歧化酶消除了半醌信号。在过氧化氢酶存在的情况下，这些成分对整体EPR谱的贡献是相等的。根据硫醇氧化和半醌形成所包含的单电子转移步骤以及硫取代和氮丙啶基损失中固有的双电子转移来讨论这些数据。
