The measurement of inorganic phosphate (Pi) is critical in biochemistry. Nearly a century ago, Fiske and Subbarow1 popularized a spectroscopic method for the determination of phosphate, based on formation of a phosphomolybdate complex. Though this method is still widely used today, it is not selective for Pi in the presence of labile phosphate compounds. If, however, Pi is precipitated as a calcium salt, as first reported by Lipmann and Tuttle,2 Pi can be separated from interfering substances before analysis. Variations of the Fiske-Subbarow method for determination of phosphate are routinely used to measure NTPase activity, but to our knowledge, the Lipmann-Tuttle modification has never been applied. Here, we present an improved assay for NTPase activity, based on the above methods, and establish its utility in measuring the activity of the ATPase nitrogenase and the GTPase elongation factor G (EF-G). ATP hydrolysis by nitrogenase is typically measured as ADP formed by HPLC, to avoid the selectivity problem of the Fiske-Subbarow method. This procedure, however, is not compatible with the ATP regeneration system added to standard nitrogenase assays, and thus provides an incomplete picture of nitrogenase reactivity. Ribosome-dependent GTP hydrolysis by EF-G is typically measured using gamma-32P-GTP. Our method is simple, less expensive, poses fewer hazards, and is at least as precise as standard methods that use HPLC and/or radiolabeled nucleotides for the measurement of nucleotide hydrolysis. Our method is especially useful in the study of nitrogenase because it is compatible with standard assay conditions, unlike many other methods for measuring nucleotide hydrolysis. References (1) Fiske, C. H.; Subbarow, Y. J. Biol. Chem. 1925, 66, 375. (2) Lipmann, F.; Tuttle, L. C. J. Biol. Chem. 1944, 153, 571.

Improved method for the spectroscopic determination of inorganic phosphate to quantify nucleotide hydrolysis

251st National Meeting (2016)

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