Iizuka T, Kanegasaki S, Makino R, Tanaka T, Ishimura Y
J Biol Chem. 1985 Oct 5;260(22):12049-53.
The b-type cytochrome in porcine neutrophils in situ was studied by the low temperature absorption spectroscopy at 77 K. Absolute spectra of the dithionite-reduced cell suspension revealed the existence of a b-type cytochrome with alpha, beta, and Soret absorption maxima at 558, 528, and 426 nm, respectively. The alpha band was unsymmetrical and showed a main peak at 558 nm with a shoulder at around 556 nm. When the cells were anaerobically stimulated either by phorbol myristate acetate or arachidonate followed by reduction by dithionite, the alpha band split clearly into double peaks at 555.5 and 558 nm, suggesting the presence of at least two states or species of the b-type cytochrome(s) in the cell. By monitoring absolute spectra of neutrophils at 77 K, we examined the possibility of CO binding to the b-type cytochrome. The absorption spectra of reduced b-type cytochrome in the presence and absence of CO, however, were not distinguishable under various conditions including equilibration with CO under high pressure or CO treatments in a dark room or at pH 8.5, 7.0, or 5.5. In contrast, the spectra of the reduced cytochrome disappeared immediately after exposure to O2, whether or not the cells had been treated with CO. The results indicate that the cytochrome does not form a CO complex in situ but reacts with O2, either directly or indirectly.
采用77K低温吸收光谱法研究了猪中性粒细胞原位b型细胞色素。连二亚硫酸盐还原的细胞悬液的绝对光谱显示存在一种b型细胞色素，其α、β和Soret吸收峰分别在558、528和426nm处。α带不对称，在558nm处有一个主峰，在556nm左右有一个肩峰。当细胞用佛波醇肉豆蔻酸酯或花生四烯酸进行厌氧刺激，随后用连二亚硫酸盐还原时，α带明显分裂为555.5和558nm处的双峰，表明细胞中至少存在两种状态或种类的b型细胞色素。通过监测77K下中性粒细胞的绝对光谱，我们研究了CO与b型细胞色素结合的可能性。然而，在包括高压下与CO平衡、在暗室中或在pH 8.5、7.0或5.5下进行CO处理等各种条件下，存在和不存在CO时还原的b型细胞色素的吸收光谱无法区分。相比之下，无论细胞是否用CO处理，还原型细胞色素的光谱在暴露于O2后立即消失。结果表明，该细胞色素在原位不形成CO复合物，而是直接或间接与O2反应。
