Rottenberg H, Moreno-Sanchez R
Pathology Department M.S. 435, Hahnemann University, Philadelphia, PA 19102.
Biochim Biophys Acta. 1993 Nov 2;1183(1):161-70. doi: 10.1016/0005-2728(93)90014-7.
A new method for the estimation of steady-state delta pH, and the rate of acidification, by H(+)-ATPases (and other proton transporters) in inverted membrane vesicles is described. The method is based on a combination of two widely used fluorescent delta pH probes, 9-aminoacridine and 9-amino-6-chloro-2-methoxyacridine. It is demonstrated that 9-amino-6-chloro-2-methoxyacridine fluorescence quenching, which is very sensitive to small pH gradients, is not sensitive to the magnitude of large pH gradient, while 9-aminoacridine, which does not sense small gradients, is very sensitive to large pH gradients. A proper mixture of the two probes provides a method which is equally sensitive to pH gradients from very small values up to 3.5 pH units. The probe response was evaluated by titrations of the fluorescence signal with nigericin and adjusted by changing the concentration ratio and the emission wavelength. In liposomes, submitochondrial particles and bacterial vesicles an almost linear dependence of quenching on delta pH over the entire range can be obtained with this method. It is demonstrated that the new method can be used to obtain more reliable estimates of the rate of acidification as well as the magnitude of delta pH, whereas each of these and similar probes, by themselves are not as reliable. A determination of the ratio delta Gp/delta muH over a wide range of values reveal that this ratio is not constant but decreases with delta Gp. This finding should be taken into consideration when attempting to estimate the H+/ATP ratio form the measurement of delta Gp/delta muH.
本文描述了一种通过H(+)-ATP酶（以及其他质子转运体）在反向膜泡中估算稳态δpH和酸化速率的新方法。该方法基于两种广泛使用的荧光δpH探针9-氨基吖啶和9-氨基-6-氯-2-甲氧基吖啶的组合。结果表明，对小pH梯度非常敏感的9-氨基-6-氯-2-甲氧基吖啶荧光猝灭对大pH梯度的大小不敏感，而对小梯度不敏感的9-氨基吖啶对大pH梯度非常敏感。两种探针的适当混合提供了一种对从非常小的值到3.5个pH单位的pH梯度同样敏感的方法。通过用尼日利亚菌素滴定荧光信号来评估探针响应，并通过改变浓度比和发射波长进行调整。用这种方法在脂质体、亚线粒体颗粒和细菌膜泡中可以得到在整个范围内猝灭对δpH几乎呈线性依赖关系。结果表明，新方法可用于更可靠地估算酸化速率以及δpH的大小，而这些探针及类似探针单独使用时并不那么可靠。在很宽的值范围内测定δGp/δμH的比值表明，该比值不是恒定的，而是随δGp降低。在试图从δGp/δμH的测量值估算H+/ATP比值时，应考虑这一发现。
