Monitoring the myosin ATPase reaction using a sensitive fluorescent probe: pyrene-labeled ATP.

A pyrene-labeled ATP (Pyr-ATP) in which a pyrene fluorophore is linked to the ribose moiety of ATP with a butyryl chain has been synthesized, together with the corresponding analog of ADP. The spectroscopic properties of two fluorescent analogs were found to be similar to those of 1-pyrenebutyric acid, making them photostable and highly sensitive probes for detecting changes in conformations around the nucleotide binding sites of proteins. Binding of Pyr-ADP to myosin subfragment-1 (S-1) resulted in a fluorescence quenching of about 70%. This binding was tight, with a dissociation constant (0.9 microM) similar to that of ADP itself. Formation of the stable ternary complex of Pyr-ADP with S-1 and orthovanadate could be monitored from the quench in pyrene fluorescence with a rate constant of 0.01 s-1. The final fluorescence intensity was about 20% of that for Pyr-ADP alone. Pyr-ATP was hydrolyzed by S-1 1.3 times faster than was ATP. Hydrolysis of Pyr-ATP was accompanied by an initial quenching of pyrene fluorescence with a subsequent recovery of the fluorescence. The fluorescence changes could be used to monitor the hydrolysis reaction continuously and measure the turnover rates of the analog. The fluorescence assay was sensitive, particularly under single turnover conditions, allowing hydrolysis reactions to be monitored at concentrations of S-1 and the analog as low as 50 nM.