8] Bioluminescent assays using coimmobilized enzymes

Abstract

This chapter describes the uses of luciferases coimmobilized with other enzymes for various analytical purposes. The quantitative determination of metabolites and enzymes is coupled to the luciferase reactions, which are measured as light production. The assays are sensitive, rapid, and specific. Two different luciferases have been used: the firefly ( Photinus pyralis ) luciferase and the bacterial ( Vibrio harveyi ) luciferase. In general, the enzymes are more stable than the soluble forms. The sensitivity of the assays is greatly increased, because of the fact that the immobilized luciferase is present in a microenvironment with locally high concentrations of ATP or FMNH2. For example, coimmobilized NADH, FMN oxidoreductase and bacterial luciferase, produce about 100 times more light per picomole of NADH than comparable amounts of soluble enzymes. Another important aspect is that immobilized enzymes can be incorporated into flow cells where they can be used for multiple assays.