Microassay with the NADH-induced light reaction, technique improved by means of purified enzymes from Achromobacter fischeri

Abstract

Purification of a commercial preparation of Achromobacter fischeri was carried out by agarose-suspension electrophoresis and by molecular-sieve chromatography. Both the luciferase and an oxidoreductase, catalyzing reduction of FMN with NADH, were obtained in more than one form. Flavins, liable to interfere with the light production in analytical applications, were present in amounts worthy of consideration, but seem to be firmly bound to protein. The major quantity was found in the enzymatically inactive fractions. In free zone electrophoresis of the main luciferase component, the mobility of the zone containing enzyme activity was calculated to -4.0 X 10(-5) cm2 sec-1 V-1 at 12 degrees C. Fractions of the two enzymes were separated and mixed in different proportions to study how the intensity and time course of NADH-induced light emission can be modified. These experiments disclosed how reaction mixtures will have to be composed in appropriate photokinetic assays, using NADH as measurable product. A regenerating system based on the purified fractions is described. Instead of the light flash, following the consumption of NADH, the light is emitted on a well maintained level, permitting assays with a less elaborate equipment than the one required for the recording of fast reactions.