ATP hydrolysis assay by ACMA quenching

With this method we indirectly measure the hydrolytic activity of the F₁F₀-ATPase by following the establishment of the resulting proton gradient, using the self-quenching property of the fluorescent dye 9-Amino-6-Chloro-2-Methoxyacridine (ACMA). The fluorescence of this membrane permeable dye quenches when a proton gradient forms across a lipid compartment⁶⁰.

The fluorescence of the dye 9-Amino-6-Chloro-2-Methoxyacridine (ACMA) (excitation wavelength: 410 nm; emission wavelength: 480 nm) was monitored with a Cary Eclipse Fluorescence Spectrophotometer from Agilent Technologies. A cuvette with stirring magnet was filled with HMK buffer (10 mM Hepes-KOH pH 7.5, 1 mM MgCl₂, 10 mM KNO₃), 150 μg/ml inverted bacterial membrane vesicles and 2 μM ACMA in a final volume of 1 ml. The fluorescence baseline, taken as 100% fluorescence to determine the percentage of quenching, was measured and hydrolysis was started by the addition of 1–500 μM ATP or ATPαS. The pH gradient established by the ATP synthase quenched the ACMA dye proportionally to the hydrolytic activity and was eventually dissipated by the addition of 20 mM NH₄Cl.

To assess the effect of valinomycin and the coupling of IMVs, a buffer containing 10 mM MOPS-KOH pH 7.5, 100 mM KCl, and 2.5 mM MgCl₂ was used instead of HMK. The measurement was carried as above, with or without 1 μM valinomycin, and started with 500 μM ATP.