Sedgwick E G, Bragg P D
Department of Biochemistry, University of British Columbia, Vancouver, Canada.
Arch Biochem Biophys. 1990 Nov 1;282(2):372-6. doi: 10.1016/0003-9861(90)90131-h.
N-Phenylnaphthylamine (NPN) has been used previously to probe the fluidity or microviscosity of membrane lipids. We have shown (Sedgwick, E. G., and Bragg, P.D. (1988) FEBS Lett. 229, 127-130) that the fluorescence intensity of this probe abruptly increases upon depletion of the oxygen content of a medium by respiring cytochrome o of Escherichia coli that has been incorporated into soybean phospholipid vesicles. We now show that the pH probes pyranine and quinacrine behave similarly to NPN. The fluorescence change is not due to changes in the pH gradient across the membrane or to a change in the distribution of probe between the vesicles and the external medium. It is insensitive to uncouplers. The fluorescence change with pyranine and quinacrine occurs also with soluble cytochrome o in the absence of added phospholipid. The NPN response requires added phospholipid. Alteration of the redox state of cytochrome o with cyanide suggests that these probes respond to a change in the redox state of the cytochrome, either by alterations in binding of the probe to the cytochrome or by a change in the environment of the probe bound to the cytochrome. This behavior should be considered when pyranine or quinacrine are used to measure changes in the internal pH of membrane vesicles containing redox proteins.
N-苯基萘胺（NPN）此前已被用于探测膜脂的流动性或微粘度。我们已经表明（塞奇威克，E.G.，和布拉格，P.D.（1988年）《欧洲生物化学学会联合会快报》229，127 - 130），当通过掺入大豆磷脂囊泡中的大肠杆菌的细胞色素o进行呼吸作用使培养基中的氧含量耗尽时，该探针的荧光强度会突然增加。我们现在表明，pH探针吡喃荧光素和喹吖因的行为与NPN相似。荧光变化不是由于跨膜pH梯度的变化或探针在囊泡与外部介质之间分布的变化所致。它对解偶联剂不敏感。在没有添加磷脂的情况下，吡喃荧光素和喹吖因的荧光变化也会与可溶性细胞色素o一起发生。NPN的响应需要添加磷脂。用氰化物改变细胞色素o的氧化还原状态表明，这些探针通过探针与细胞色素结合的改变或与细胞色素结合的探针环境的变化，对细胞色素氧化还原状态的变化做出反应。当使用吡喃荧光素或喹吖因来测量含有氧化还原蛋白的膜囊泡内部pH的变化时，应考虑这种行为。
