The proton pumping activity of H(+)-ATPases: an improved fluorescence assay

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.