Real-Time Colorimetric Assay of Inorganic Pyrophosphatase Activity Based on Reversibly Competitive Coordination of Cu2+ between Cysteine and Pyrophosphate Ion

Abstract

In this study we demonstrate a new colorimetric method for real-time pyrophosphatase (PPase) activity assay based on reversible tuning of the dispersion/aggregation states of gold nanoparticles (Au-NPs) by controlling the coordination of Cu(2+) between cysteine and pyrophosphate ion (PPi) with PPase. The addition of Cu(2+) to the cysteine-stabilized Au-NP dispersion results in the aggregation of Au-NPs, while the further addition of PPi to this aggregation turns the aggregated Au-NPs into their dispersed state because of the higher coordination reactivity between Cu(2+) and PPi than that between Cu(2+) and cysteine. The subsequent addition of PPase to the PPi-triggered dispersed Au-NPs restores the aggregation state of Au-NPs because PPase catalyzes the hydrolysis of PPi into orthophosphate and thus consumes PPi in the reaction system. In this study, we utilize this reversibility of the change between the aggregation/dispersion states of Au-NPs for real-time colorimetric monitoring of PPase activity by continuously measuring the ratio of absorbance at the wavelength of 650 nm (A650) to that at 522 nm (A522) in the time-dependent UV-vis spectra of Au-NP dispersions containing different activities of PPase. To calculate the kinetics of the PPase-catalyzed hydrolysis of PPi, the A650/A522 values are converted into PPi concentrations to obtain the time-dependent changes of PPi concentrations in the dispersions containing different activities of PPase. The initial reaction rates (v0) are thus achieved from the time-dependent logarithm of PPi concentrations with the presence of different PPase activities. Under the experimental conditions employed here, the v0 values are linear with the PPase activity within a range from 0.025 to 0.4 U with a detection limit down to 0.010 U (S/N = 3). Moreover, the colorimetric method developed here is also employed for PPase inhibitor evaluation. This study offers a simple yet effective method for real-time PPase activity assay.