REACTIONS OF HALOGEN-SUBSTITUTED AZIRIDINYLBENZOQUINONES WITH GLUTATHIONE - FORMATION OF DIGLUTATHIONYL CONJUGATES AND SEMIQUINONES

The reaction between glutathione and 2,5-diaziridinyl-1,4-benzoquinone s bearing halogen substituents at C-3 and C-6 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 t he halogen atoms toward sulfur substitution is the primary reaction le ading to the formation of mono-and di-glutathionyl-substituted quinone s. The relative formation of these conjugates depended on the GSH/quin one molar ratios. At GSH/quinone molar ratios below unity, the product s 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 (a(N) = 1.98 G) and assigned to the semiquinone form of dichloro-diaziridinyl benzoquinone. 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/qui none molar ratios above unity (4, 10 and 100 molar excess of GSH) a pa ttern of products emerged consisting of 3,6-diglutathionyl quinones wi th two, one and no aziridinyl moieties, identified by mass spectral an alysis. EPR studies revealed that these compounds were minor component s of a composite EPR spectrum (a 3-line signal with 1:1:1 relative int ensities, 1 equivalent N (a(N) = 1.73 G) and 1 H (a(H) = 1.45 G) or a 3-line signal with 1:2:1 relative intensities and 2 equivalent H (a(H) = 1.4 G). These minor components were assigned to the diglutathionyl conjugates bearing one-or no aziridinyl moiety, respectively. The majo r component in the EPR signal showed a 3-line spectrum (1:1:1 relative intensity) with 1 equivalent N (a(N) = 1.7 G) and a g shift of -0.96 G. This spectrum was assigned to a triglutathionyl conjugate of a mono aziridinylbenzoquinone. This major component was also observed when GS H/quinone mixtures were incubated with the two-electron transfer flavo protein NAD(P)H:quinone oxidoreductase. The semiquinone signals were a bolished by superoxide dismutase. In the presence of catalase, the con tribution of these components to the overall EPR spectrum was equal. T hese data are discussed in terms of the one-electron transfer steps en compassed by thiol oxidation and semiquinone formation and the two-ele ctron transfers inherent in sulfur substitution and aziridinyl group l oss. (C) 1998 Elsevier Science Ireland Ltd. All rights reserved.