Govindjee R, Ebrey T G, Crofts A R
Biophys J. 1980 May;30(2):231-42. doi: 10.1016/S0006-3495(80)85091-0.
The quantum yield of H+ release in purple membrane (PM) sheets, and H+ uptake in phospholipid (egg phosphatidylcholine, PC) vesicles containing PM, was measured in single turnover light flashes using a pH-sensitive dye, p-nitrophenol, with rhodopsin as an actinometer. We have also calculated the ratio of H+ released per M412 formed (an unprotonated Shiff-base intermediate formed during the photocycle). In PM sheets, the quantum yield of H+ release depends on the medium. The quantum yield of M412 is independent of salt concentration. The ratio H+/M412 is approximately 1.8 M KC; and approximately 0.64 in 10 mM KCl. Direct measurements of the quantum yield of H+ give approximately 0.7 when the PM is suspended in 0.5 M KC; and 0.25 in 10 mM KCl. Using a quantum yield for M412 formation of 0.3 (Becher and Ebrey, 1977 Biophys J. 17:185.), these measurements also give a H+/M412 approximately 2 at high salt. In PM/PC vesicles, the H+/M412 is approximately 2 at all salt concentrations. The M412 decay is biphasic and the dye absorption change is monophasic. The dissipation of the proton gradient is very slow, taking on the order of seconds. Addition of nigericin (H+/K+ antiporter) drastically reduces the pH changes observed in PM/PC vesicles. This and the observation that the proton relaxation time is much longer than the photochemical cycling time suggest that the protons are pumped across the membrane and there is no contribution as a result of reversible binding and release of protons on just one side of the membrane.
使用对pH敏感的染料对硝基苯酚，并以视紫红质作为光度计，通过单次周转光脉冲测量了紫色膜（PM）片层中H⁺的释放量子产率，以及含有PM的磷脂（鸡蛋磷脂酰胆碱，PC）囊泡中H⁺的摄取。我们还计算了每形成一个M412（光循环过程中形成的未质子化席夫碱中间体）释放的H⁺的比率。在PM片层中，H⁺释放的量子产率取决于介质。M412的量子产率与盐浓度无关。H⁺/M412的比率在1.8 M KCl中约为1.8；在10 mM KCl中约为0.64。当PM悬浮在0.5 M KCl中时，对H⁺量子产率的直接测量约为0.7；在10 mM KCl中为0.25。使用M412形成的量子产率为0.3（Becher和Ebrey，1977年，《生物物理杂志》17:185），这些测量在高盐条件下也得出H⁺/M412约为2。在PM/PC囊泡中，在所有盐浓度下H⁺/M412约为2。M412的衰减是双相的，而染料吸收变化是单相的。质子梯度的耗散非常缓慢，大约需要几秒钟。加入尼日利亚菌素（H⁺/K⁺反向转运体）会显著降低在PM/PC囊泡中观察到的pH变化。这以及质子弛豫时间比光化学循环时间长得多的观察结果表明，质子被泵过膜，并且不存在由于质子在膜一侧可逆结合和释放而产生的贡献。
