Inorganic pyrophosphate (PPi) and higher polyphosphates were first recognized in biological systems as constituents of yeast, and other fungi (1). Subsequent work showed PPi to be involved in the biosynthesis and transformation of a wide variety of biological substances (Z-4,9). Recently PPi in addition to adenosine triphosphate (ATP) was found to be a product of photophosphorylation in chromatophores of Rhodospirillum rubrum (5,6).
Our interest in the mechanism of energy conservation in R. rubrum (10,13) required that we be able to estimate PPi in the presence of a five-to ten-fold higher concentration of ATP. The usual method for PPi analysis, which involves hydrolysis of PPi followed by the determination of inorganic orthophosphate, is inadequate for this purpose because of the concomitant hydrolysis of the pyrophosphate bonds of ATP. In addition, we required a system which would rapidly convert PPi to a stable product not susceptible to a highly active pyrophosphatase enzyme present in the 11. rubrum membrane (10,23).
In submitochondrial particles, maximum rates of aerobic ATP synthesis from ADP and Pi can be measured only when the terminal phosphate of ,4TP is trapped in a metabolically stable form. The classical trapping system used for mitochondrial oxidative phosphorylation consists of the enzyme hexokinase together with its substrate glucose. In the presence