A delta pH clamp method for analysis of steady-state kinetics of photophosphorylation

An instrumental device is described which allows steady-state kinetic measurements of photophosphorylation at a desired proton gradient which can be maintained throughout the course of the experiment ('delta pH clamp'). This is achieved by electronic regulation of light intensity using the calibrated 9-aminoacridine fluorescence signal as sensor of the gradient. The instrument is suitable for determination of kinetic parameters of the proton-translocating ATPase in isolated envelope-free chloroplasts under defined conditions. At clamped delta pH, phosphorylation as a function of substrate concentration shows Michaelis-Menten kinetics. The true Michaelis constants and the dissociation constants for phosphate and ADP are reported. The Michaelis constants are not affected by the magnitude of the proton gradient in the investigated range. The significance of these results is discussed.