Methodological aspects on the firefly luciferase assay of adenine nucleotides in whole blood and red blood cells

Abstract

The firefly luciferase assay of adenosine-5'-triphosphate (ATP) is used in a simple procedure for the determination of adenine nucleotides in whole blood and red blood cells. Cells are lysed with cold trichloroacetic acid and nucleotides are determined in the lysate after dilution with buffer. In a comparative study with the spectrophotometric assay of ATP, extracts were prepared by centrifugation of the lysate to remove interfering turbidity. This step resulted in a significant loss of ATP in the clear supernatant. This loss could be avoided by determination of ATP by the firefly luciferase assay in diluted uncentrifuged lysates. The yields of ATP, ATP + ADP and ATP + ADP + AMP were compared in a red blood cell preparation after lysis with trichloroacetic acid and another lytic method based on the detergent triton X-100. The precision was better with acid whereas yields were essentially the same with both methods. The method of standardization, external or internal, was of little practical importance regarding yields and precision, but internal calibration is recommended to ensure that there is no interaction of the luciferase reaction with lytic reagent or sample.