THE PROTON-PUMPING ACTIVITY OF H-ATPASES - AN IMPROVED FLUORESCENCE ASSAY()

A new method for the estimation of steady-state DELTApH, and the rate of acidification, by H+-ATPases (and other proton transporters) in inv erted membrane vesicles is described. The method is based on a combina tion of two widely used fluorescent DELTApH probes, 9-aminoacridine an d 9-amino-6-chloro-2-methoxyacridine. It is demonstrated that 9-amino- 6-chloro-2-methoxyacridine fluorescence quenching, which is very sensi tive to small pH gradients, is not sensitive to the magnitude of large pH gradient, while 9-aminoacridine, which does not sense small gradie nts, is very sensitive to large pH gradients. A proper mixture of the two probes provides a method which is equally sensitive to pH gradient s from very small values up to 3.5 pH units. The probe response was ev aluated by titrations of the fluorescence signal with nigericin and ad justed by changing the concentration ratio and the emission wavelength . In liposomes, submitochondrial particles and bacterial vesicles an a lmost linear dependence of quenching on DELTApH over the entire range can be obtained with this method. It is demonstrated that the new meth od can be used to obtain more reliable estimates of the rate of acidif ication as well as the magnitude of DELTApH, whereas each of these and similar probes, by themselves are not as reliable. A determination of the ratio DELTAG(p)/DELTAmu(H) over a wide range of values reveal tha t this ratio is not constant but decreases with DELTAG(p). This findin g should be taken into consideration when attempting to estimate the H +/ATP ratio form the measurement of DELTAG(p)/DELTAmu(H).