On the role of citrate in 12-molybdophosphoric-acid methods for quantification of phosphate in the presence of ATP†
ATP plays a central role in life processes, where a large number of specific enzymes catalyse its hydrolysis. A critical step in the evaluation of the catalytic activity of these enzymes is the analytical determination of the release of phosphate anions in the presence of ATP. Citrate has been proposed as a useful additive for measuring these activities avoiding the interference of phosphate release after catalysis. However, the underlying mechanism remains unclear. Here we explore this effect using the malachite green procedure for determination of phosphate as a sensor. First, we evaluate the kinetics of formation of the 12-molybdophosphoric acid–malachite green complex at different temperatures and found that at least two intermediate species, with distinctive spectroscopic properties, and a final reversible step are needed to account for the experimental results. Addition of citrate produces a decrease in the rate of formation of the colour species without a significant effect on the dissociation of the preformed complexes. For the kinetic analysis, citrate is introduced in this mechanism by forming two complexes with free molybdate without affecting the other reaction steps. A lag phase in formation of colour species becomes evident at high citrate-to-molybdate mole ratios, which is extended for at least 8 h at a mole ratio of 4 : 1. This allows, when measuring the enzymatic hydrolysis of ATP, the small amount of phosphate generated by non-enzyme-catalyzed hydrolysis after the enzymatic reaction is stopped, not produce a significant change in the measured signal.