The identity and properties of firefly luciferase inhibitors in urine

Abstract

The firefly luciferin-luciferase reagent may be used for the determination of low concentrations of adenosine 5′-triphosphate (ATP). The lower limit of ATP measurements in body fluids is dependent upon the concentrations of inhibitors; for example, chloride, sulfate, urea. For urine and for a mixture of chloride, sulfate, and urea the peak light output, following mixing of luciferase, luciferin, and ATP, decreased exponentially with increasing inhibitor concentration. On the other hand peak light output increases linearly with increasing ATP concentration. We have established a mathematical relationship between the exponential inhibitor response and the linear substrate response during codilution. This relationship predicted the existence of a dilution for each initial ATP-inhibitor mixture which would yield maximum light output. Such predictions agreed qualitatively with experimental observations when ATP-urine mixtures were diluted.