A microprocedure for the colorimetric determination of inorganic pyrophosphate (PP0 in the presence or absence of orthophosphate (Pi) has been developed. PPi is estimated quantitatively as the amount of chromophore formed with molybdate reagent, 1-amino-2-naphthol-4-sulfonic acid in bisulfite and thiol reagent (monothioglycerol or 2-mercaptoethanol). The latter is obligatory for color formation. P~ is estimated without thiol reagent. The two chromophores differ in absorption spectra, the greatest difference being at 580 nm. For both, color develops fully by 10 min and is stable up to 1 hr. Just less than 0.4 IxM PPi can be determined. The extinction coefficients are 2.70 Γ 104 and 8.76 x 103 for PPi and Pi, respectively, both with thiol reagent present, and 2.77 Γ 103 for Pi with no thiol reagent.
A ten-fold excess of P~ does not interfere with the determination of PPI and in fact can be estimated in the same mixture. A 15-fold excess, however, diminishes the accuracy of PP~ estimations. Trichloroacetic acid and sodium fluoride inhibit color formation, but this inhibition is overcome by the addition of sodium acetate buffer, pH 4.0. Nucleoside triphosphates and adenosine 3':5'-cyclic monophosphate are stable in the reaction mixture.
The method was tested in assays of Escherichia coli DNA-dependent RNA polymerase (nucleoside triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6). Progress curves measured by either the rate of PP~ formation or the rate of synthesis of labeled RNA were very similar. Product PPi formed by as little as 0.6 unit of RNA polymerase in a 225qxl incubation medium could be measured.
An automated version of the method was devised which allows accurate determination of PP~ down to 1 IXN (without range expander attachment) at a sampling rate of 20-40 tubes/hr.
Earlier, the usual procedure for the colorimetric analysis of PPi involved hydrolyzing it in acid (1) prior to determination of the resulting Pi by the molybdenum blue method of Fiske and Subbarow (2). Later, Flynn et al. (3) introduced a procedure to measure the molybdate-PP~ complex that is slowly reduced by cysteine (4). P~ interfered and, because of the lengthy period of color development, so did labile organic