Colorimetric determination of inorganic pyrophosphate by a manual or automated method.

A microprocedure for the colorimetric determination of inorganic pyrophosphate (PP_i) in the presence or absence of orthophosphate (P_i) has been developed. PP_i 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_i 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 μm PP_i can be detemined. The extinction coefficients are 2.70 × 10⁴ and 8.76 × 10³ for PP_i and P_i, respectively, both with thiol reagent present, and 2.77 × 10³ for P_i with no thiol reagent.A ten-fold excess of P_i does not interfere with the determination of PP_i and in fact can be estimated in the same mixture. A 15-fold excess, however, diminishes the accuracy of PP_i estimations. Trichloroacetic acid and sodium fluoride inhibi 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_i formation or the rate of synthesis of labeled RNA were very similar. Product PP_i formed by as little as 0.6 unit of RNA polymerase in a 225-μl incubation medium could be measured.An automated version of the method was devised which allows accurate determination of PP_i down to 1 μm (without range expander attachment) at a sampling rate of 20–40 tubes/hr.