A colorimetric determination of inorganic pyrophosphate.

During recent work on the mechanism of the ATP-CoA-acetate1 reaction (1, 2) we observed that, on incubation of yeast enzyme with ATP, CoA, acetate, and hydroxylamine, a substance was formed in the presence of fluoride which, although initially colorless with the Fiske-Subbarow phosphate reagents (3), slowly developed on standing a blue color of a somewhat more purplish shade than that obtained with orthophosphate. This peculiar color reaction reminded us of an earlier observation of Davenport and Sacks (4), who described a similar color reaction due to inorganic pyrophosphate. The observation of the slow color development was an important step toward the identification of pyrophosphate as the product of the ATP-CoA-acetate reaction. This prompted us to attempt a use of this rather specific effect of inorganic pyrophosphat’e for its quantitative det’ermination. During our efforts to work out such a method we became aware of considerable difference in the rate of coloring with different samples of the aminonaphtholsulfonic acid-sulfite-bisulfite reagent (3). It appeared that freshly prepared solutions showed very sluggish reaction, while aged solutions frequently, but rather inconsistently, gave a more rapid coloring. It was eventually found that the rate of color formation could be enhanced and stabilized by the addition of cysteine, glutathione, or other sulfhydryl compounds, including hydrogen sulfide. Hydrosulfite is also active and ascorbic acid has a slight but appreciable effect. By addition of the sulfhydryl compound the rather erratic behavior of ordinary reagents could be eliminated and, for practical purposes, cysteine was chosen as a most convenient accelerator. Addition of cysteine does not affect the color due to orthophosphate. The effect of various concentrations of cysteine on the color produced by a standard amount of 0.5 pmole of pyrophosphate is presented in Fig. 1, from