Firefly and bacterial luminescence: basic science and applications.

The basic chemistry of the reactions leading to light emission in the firefly and in bacteria are briefly reviewed. With excess firefly reagents, the light intensity is proportional to the ATP concentration. For this reason, the reagents have been used for ATP determination in a number of important biological systems. A number of such applications are reviewed. With excess bacterial reagents, the light intensity is directly proportional to the reduced pyridine nucleotide concentration (NADH or NADPH). The applications of this system for studying reactions involving dehydrogenases using NAD or NADP as electron acceptors are presented. Many assays have now been developed using enzymes immobilized on Sepharose. The advantages of using the immobilized enzymes are greater stability of the immobilized enzymes over the soluble forms; increased sensitivity of detection relative to the soluble forms, and reusability of the immobilized enzymes. A comparison of the immobilized bioluminescent assay for 7 alpha-hydroxysteroid with gas-liquid chromatography and radioimmunoassay is presented. Coimmobilized enzymes can be packed in a flow cell and used in an automated instrument with good reproducibility. It is likely that future developments of bioluminescent assays for ATP or NAD(P)H will be with immobilized enzymes using an automated instrument.