The photometric estimation of luciferase in luminous bacterial cells.

Recent studies have shown that t,he luminescence of luminous bacteria under certain conditions responds to ionizing radiation instantaneously (1) and, under other conditions, responds to doses as low as 500 rads (2). The intensity of the luminescence is proportional to the rate of th(, luciferase reaction taking place in viva. Thus radiation effects both on the metabolic system supporting the luciferase reaction (3) and on the enzyme itself may contribute to the observed response. In order to evaluate the relative contributions of these two actions, it is necessary to establish a method for the assay of active luciferase in the cells. This paper describes an assay based on measurement of the luminous intensity of the luciferase reaction. Applications of this method will be found for irradiated cells (3) and for growing cells (4). The procedure is derived from the work of Strchler and Cormier (5 1, McElroy et al. (6), and Strehler (7), who identified the reaction components required for maximum luminescence in cell-free extracts of Photobacterium, fischeri. These components are reduced riboflavin phosphate (reduced Aavin mononucleotide, FMNH,), palmitic or other homologous aldehydc (5)) oxygen, and the bacterial extract (luciferase). The cell-free extracts also contain an FMN-linked diphosphopyridine nucleotide (DPNH) oxidase which permits the substitution of a steadystate FMXH,-generating system for the spontaneously autoxidizable FMNH,. Nothing presently understood about the mechanism of the luciferase reaction suggests a method for measuring luciferase activity on a chemical basis. The extracts referred to above were prepared by lysing the cells in deionized water and removing the cell debris by highspeed centrifugation to obtain an optically clear solution. The present assay was based on these crude extracts because no method of purification exists which permits the necessary total recovery of enzyme. When such extracts are