Zhou J S, Hoffman B M
Department of Chemistry, Northwestern University, Evanston, IL 60208-3113.
Science. 1994 Sep 16;265(5179):1693-6. doi: 10.1126/science.8085152.
A reverse protocol for measurements of molecular binding and reactivity by excited-state quenching has been developed in which the quencher, held at a fixed concentration, is titrated by a photoexcitable probe molecule whose decay is monitored. The binding stoichiometries, affinities, and reactivities of the electron-transfer complexes between cytochrome c (Cc) and cytochrome c peroxidase (CcP) were determined over a wide range of ionic strengths (4.5 to 118 millimolar) by the study of photoinduced electron-transfer quenching of the triplet excited state of zinc-substituted Cc (ZnCc) by Fe3+CcP. The 2:1 stoichiometry seen for the binding of Cc to CcP at low ionic strength persists at the physiologically relevant ionic strengths and likely has functional significance. Analysis of the stoichiometric binding and rate constants confirms that one surface domain of CcP binds Cc with a high affinity but with poor electron-transfer quenching of triplet-state ZnCc, whereas a second binds weakly but with a high rate of electron-transfer quenching.
已开发出一种通过激发态猝灭测量分子结合和反应性的反向方案，其中固定浓度的猝灭剂由光激发探针分子滴定，同时监测该探针分子的衰变。通过研究铁离子细胞色素c过氧化物酶（Fe3+CcP）对锌取代细胞色素c（ZnCc）三重激发态的光诱导电子转移猝灭，在很宽的离子强度范围（4.5至118毫摩尔）内测定了细胞色素c（Cc）与细胞色素c过氧化物酶（CcP）之间电子转移复合物的结合化学计量、亲和力和反应性。在低离子强度下观察到的Cc与CcP结合的2:1化学计量在生理相关离子强度下依然存在，并且可能具有功能意义。对化学计量结合和速率常数的分析证实，CcP的一个表面结构域以高亲和力结合Cc，但对三重态ZnCc的电子转移猝灭能力较差，而另一个结构域结合较弱，但电子转移猝灭速率较高。
