Orii Y, Miki T, Kakinuma K
Department of Public Health, Faculty of Medicine, Kyoto University, Japan.
Photochem Photobiol. 1995 Mar;61(3):261-8. doi: 10.1111/j.1751-1097.1995.tb03969.x.
Cytochrome b558 in solubilized membranes prepared from porcine neutrophils was reduced by dithionite with a second-order rate constant of 2.5 x 10(6) M-1 s-1 at pH 7.4 and 20 degrees C accompanied by spectral changes with peaks at 428 nm and 560 nm and isosbestic points at 420 and 441 nm. When an anaerobic mixture of solubilized membranes and NAD(P)H was exposed to a white light, cytochrome b558 was reduced biphasically but with almost the same spectral profiles as in the dithionite reduction. Thus, participation of redox component(s) of unknown nature in the photochemical reduction was suggested. The NAD(P). radical generated by photoexcitation of NAD(P)H with a 355 nm laser pulse under anaerobic conditions also reduced cytochrome b558 with a high rate constant of 4.3 x 10(8) M-1 s-1 at pH 7.4 and 20 degrees C. The reduction of cytochrome b558 accompanied a simultaneous reduction of a component having an absorption band around 420 nm, suggesting participation of an iron-sulfur (Fe-S) cluster. The cytochrome b558 reduction was followed by its reoxidation by another component with an apparent second-order rate constant of 6.5 x 10(5) M-1 s-1. During the reoxidation, the Fe-S-like component remained in the reduced state, and thus its role other than as electron mediator in neutrophils NADPH oxidase is suggested. Not only the rate constant but also the extent of cytochrome b558 reoxidation decreased as the same reaction mixture was exposed to the laser pulse repeatedly. This result clearly indicates that an electron accumulates in this electron-accepting component designated tentatively as the omega component.
用连二亚硫酸盐还原从猪嗜中性粒细胞制备的溶解膜中的细胞色素b558，在pH 7.4和20℃时二级速率常数为2.5×10⁶ M⁻¹ s⁻¹，同时伴有光谱变化，峰值在428 nm和560 nm，等吸收点在420和441 nm。当溶解膜与NAD(P)H的厌氧混合物暴露于白光下时，细胞色素b558呈双相还原，但光谱特征与连二亚硫酸盐还原时几乎相同。因此，提示有未知性质的氧化还原成分参与了光化学还原。在厌氧条件下用355 nm激光脉冲光激发NAD(P)H产生的NAD(P)·自由基也能以4.3×10⁸ M⁻¹ s⁻¹的高速率常数在pH 7.4和20℃时还原细胞色素b558。细胞色素b558的还原伴随着一个在420 nm左右有吸收带的成分的同时还原，提示有铁硫（Fe-S）簇参与。细胞色素b558还原后又被另一种成分再氧化，表观二级速率常数为6.5×10⁵ M⁻¹ s⁻¹。在再氧化过程中，类Fe-S成分保持还原状态，因此提示其在嗜中性粒细胞NADPH氧化酶中除作为电子介质外还有其他作用。随着同一反应混合物反复暴露于激光脉冲下，不仅细胞色素b558再氧化的速率常数而且其程度都降低。这一结果清楚地表明电子在这个暂定为ω成分的电子接受成分中积累。
