Kinetics of electron transfer between cytochromes c' and the semiquinones of free flavin and clostridial flavodoxin.
Kinetics of electron transfer between cytochromes c' and the semiquinones of free flavin and clostridial flavodoxin.
复制标题
细胞色素 c 与游离黄素和梭菌黄素氧还蛋白的半醌之间的电子转移动力学。
DOI：
10.1021/bi00354a029
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发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Tollin,G
中科院分区：
文献类型：
--
作者：
Meyer,TE;Cheddar,G;Bartsch,RG;Getzoff,ED;Cusanovich,MA;Tollin,G
材料和方法激光闪光光解和停流技术以及数据收集和分析方法如前所述(Ahmad等人，1981；Simondsen&Tollin等人，1983；Meyer等人，1983；Tollin等人，1984；Przysiecki等人，1985)。在所有情况下，根据Bartsch(1978)的一般程序制备纯蛋白质。在3-4个半衰期的厌氧条件下，高自旋蛋白(细胞色素co)在620 nm处出现假一级黄素衰变，低自旋蛋白(细胞色素c-556和c-554)在585 nm处出现还原细胞色素。蛋白质浓度在5-10倍的范围内变化，最小为-15微米，最大为-150微米。通常使用四到五个浓度来确定二级常数。未观察到饱和效应。FMN是通过Bio-Gel P-2凝胶过滤柱与双蒸水平衡(Nagy等人，1982)并冷冻干燥得到的。解决方案是在使用之前制作的。标准缓冲液包括40 GM黄素、10 mM EDTA和20 mM磷酸二氢钠，pH 7.0，用于光黄素和核黄素实验。用于FMN实验的16 mm离子强度缓冲液包含1 mm EDTA和5 mm磷酸盐；60 mm离子强度缓冲液包含5 mm EDTA和16 mm磷酸盐。较高的离子强度由10 mM EDTA和20、52、100和222 mM磷酸盐组成，pH为7.0，离子强度分别为96、160、256和500 mm。
Materials and MethodsThe laser flash photolysis and stopped-flow techniques and the methods of data collection and analysis were as described previously (Ahmad et al., 1981; Simondsen & Tollin, 1983; Meyer et al., 1983, 1984; Tollin et al., 1984; Przysiecki et al., 1985). In all cases, pure proteins were prepared according to the general procedures of Bartsch (1978). The pseudo-first-order decay of the flavin semiquinoneand the appearance of the reduced cytochrome were monitored at 620 nm for the high-spin proteins (cytochromes cO and at 585 nm for the low-spin proteins (cytochromes c-556and c-554) under anaerobic conditions over 3-4 half-lives. Protein concentrations were varied over a 5-10-fold range, minimally at—15, uM and maximally at— 150/uM. Typically four to five concen-trations were used to determine the second-orderrate con-stants. No saturation effects were observed. FMN was pu-rified by passage through a Bio-Gel P-2 gel filtration column equilibrated with double-distilled water (Nagy et al., 1982) and lyophilized. Solutions were made up just prior to use. Standard buffers contained 40 gM flavin, 10 mM EDTA, and 20 mM sodium phosphate, pH 7.0, for lumiflavin and riboflavin experiments. The 16 mM ionic strength buffer for the FMN experiments contained 1 mM EDTA and 5 mM phosphate; the 60 mM ionicstrength buffer contained 5 mM EDTA and 16 mM phosphate. Higher ionic strengths were made up with 10 mM EDTA and 20, 52, 100, and 222 mM phosphate, pH 7.0, giving ionic strengths of 96, 160, 256, and 500 mM, respectively.