An enzymatic cycling method using pyruvate orthophosphate dikinase and firefly luciferase for the simultaneous determination of ATP and AMP (RNA)

A novel bioluminescent enzymatic cycling assay for ATP and AMP with concomi tant use of firefly luciferase and pyruvate orthophosphate dikinase (PPDK) was developed. In this system, AMP and pyrophosphate produced from ATP by f irefly luciferase were converted back into ATP by PPDK. This resulted in co nstant luminescence once the stable phase had been reached. Background lumi nescence of the reagent was reduced with adenosine phosphate deaminase by d egrading ATP and AMP in the reagent. The maximum recycling ratio calculated from the integrated luminescence value was 2.64 cycles/min. The measurable ranges for ATP and AMP were equal and were between 4 x 10(-13) and 4 x 10( -17) mol/assay. The amount of yeast RNA could be estimated in the range of 1 x 10(-8) to 1 x 10(-12) g/assay by estimating the amount of AMP resulting from the degradation of RNA with nuclease P1. Various food samples were su bjected to measurement of the amount of ATP + AMP + RNA to provide an index for hygiene monitoring. For beef extract, sensitivity was improved by more than 20 million compared to the previous methods relying only on the amoun t of ATP as an index. (C) 1999 Academic Press.