Detection of ATP and NADH: A bioluminescent experience

Abstract

Everyone who has seen the flash of a firefly has observed the phenomenon of bioluminescence. Biological light production in various animals, plants, and bacteria has only recently been understood in molecular terms. Light production is dependent upon ATP in firefly tails (1) and reduced coenzymes such as NADH or FMNH2 in some bacteria (2). These findings form the basis for using bioluminescence as a sensitive analytical tool for detecting extremely small quantities of ATP and NADH down to 10"15 and 10"14 moles, respectively (3,4). Bioluminescent methods have wide-ranging utility for analysis; they have been used in detection of bacterial contamination, in determination of cell viability, in developing sensitive immunochemical assays, and in research areas where sensitive enzyme assays are required (3,5).Described below is a bioluminescent assay for ATP and NADH that meets the needs of an undergraduate biochemistry laboratory course. This experiment provides students with experience in both bioluminescence and analytical biochemistry yet requires limited instrumentation, preparation, and expense. The experiment as described can be run in one 3-hr time period and has potential for being used in conjunction with the measurement of metabolic levels of nucleotides and coenzymes.

Bringing Light to Science Undergraduate Students: A Successful Laboratory Experiment Illustrating the Principles and Applications of Bioluminescence

“…In spite of the aforementioned relevance of bioluminescence-based analytical methods, their application to the laboratory training of science undergraduate students is uncommon. Although some laboratory experiments have been designed to teach chemoluminescence, often resulting from the luminol reaction, most of them are demonstrations or do not make use of a luminometer. − As for bioluminescence, there are very few articles for education purposes, most of them related to either the enzymatic characterization of luciferase or its use as a reporter enzyme. − Nevertheless, the reported laboratory experiments aimed to highlight the use in the analytical lab of the luciferase reaction are very scarce . With the main objective of contributing to fill this gap, we have implemented at the University of Malaga (Spain) a laboratory experiment that, aiming at the evaluation of microbial contaminations in water, illustrates in the framework of enzymatic analysis the principles and applications of bioluminescence to science undergraduate students.…”

“…14−18 Nevertheless, the reported laboratory experiments aimed to highlight the use in the analytical lab of the luciferase reaction are very scarce. 19 With the main objective of contributing to fill this gap, we have implemented at the University of Malaga (Spain) a laboratory experiment that, aiming at the evaluation of microbial contaminations in water, illustrates in the framework of enzymatic analysis the principles and applications of bioluminescence to science undergraduate students. By using an affordable commercial reagent, bacteria are lysed and their ATP released and then quantified by means of the reaction catalyzed by a recombinant thermostable luciferase.…”