Vitello L B, Erman J E
Department of Chemistry, Northern Illinois University, DeKalb 60115.
Arch Biochem Biophys. 1987 Nov 1;258(2):621-9. doi: 10.1016/0003-9861(87)90385-7.
The binding of horse heart cytochrome c to yeast cytochrome c peroxidase in which the heme group was replaced by protoporphyrin IX was determined by a fluorescence quenching technique. The association between ferricytochrome c and cytochrome c peroxidase was investigated at pH 6.0 in cacodylate/KNO3 buffers. Ionic strength was varied between 3.5 mM and 1.0 M. No binding occurs at 1.0 M ionic strength although there was a substantial decrease in fluorescence intensity due to the inner filter effect. After correcting for the inner filter effect, significant quenching of porphyrin cytochrome c peroxidase fluorescence by ferricytochrome c was observed at 0.1 M ionic strength and below. The quenching could be described by 1:1 complex formation between the two proteins. Values of the equilibrium dissociation constant determined from the fluorescence quenching data are in excellent agreement with those determined previously for the native enzyme-ferricytochrome c complex at pH 6.0 by difference spectrophotometry (J. E. Erman and L. B. Vitello (1980) J. Biol. Chem. 225, 6224-6227). The binding of both ferri- and ferrocytochrome c to cytochrome c peroxidase was investigated at pH 7.5 as functions of ionic strength in phosphate/KNO3 buffers using the fluorescence quenching technique. The binding in independent of the redox state of cytochrome c between 10 and 20 mM ionic strength, but ferricytochrome c binds with greater affinity at 30 mM ionic strength and above.
采用荧光猝灭技术测定了马心细胞色素c与酵母细胞色素c过氧化物酶（其中血红素基团被原卟啉IX取代）的结合情况。在pH 6.0的二甲胂酸钾/KNO₃缓冲液中研究了高铁细胞色素c与细胞色素c过氧化物酶之间的缔合。离子强度在3.5 mM至1.0 M之间变化。在1.0 M离子强度下未发生结合，尽管由于内滤效应荧光强度有显著降低。校正内滤效应后，在0.1 M及以下离子强度下观察到高铁细胞色素c对卟啉细胞色素c过氧化物酶荧光有显著猝灭。这种猝灭可用两种蛋白质之间形成1:1复合物来描述。由荧光猝灭数据确定的平衡解离常数的值与先前通过差示分光光度法在pH 6.0下测定的天然酶 - 高铁细胞色素c复合物的值非常一致（J. E. 埃尔曼和L. B. 维泰洛（1980年）《生物化学杂志》225卷，6224 - 6227页）。使用荧光猝灭技术在pH 7.5的磷酸盐/KNO₃缓冲液中研究了高铁和亚铁细胞色素c与细胞色素c过氧化物酶的结合情况，作为离子强度的函数。在10至20 mM离子强度之间，结合与细胞色素c的氧化还原状态无关，但在30 mM及以上离子强度下，高铁细胞色素c结合亲和力更高。
