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Comparative studies on different bacterial luciferase preparations

1956; Elsevier BV; Volume: 63; Issue: 2 Linguagem: Inglês

10.1016/0003-9861(56)90056-x

1096-0384

Milton J. Cormier, John R. Totter, H. H. Rostorfer,

Photoreceptor and optogenetics research

Various bacterial luciferase preparations have been compared for their ability to oxidize DPNH in the presence of quinones and ferricyanide and for their ability to oxidize DPNH in the presence of FMN, longchain aldehydes, and O2 for light production. From such studies it was concluded that luciferase is not a DPNH oxidase and that the only apparent function of the enzyme is that of light production. Purification of five different types of luciferase preparations by starch-column electrophoresis resulted in an enzyme which would give light in the presence of DPNH, FMN, long-chain aldehydes, and O2 only when DPNH oxidase obtained from E. coli was added. Some luciferase preparations from A. fischeri showed an absolute requirement for albumin before light was emitted in the presence of FMNH2 and aldehyde, whereas others gave light in the absence of albumin. Cysteine, glutathione, Versene, and BAL were quite effective in replacing albumin. Apparently, the nature of the activation of purified luciferase by these compounds lies, for the most part, in the removal of heavy metals, which are known to inhibit bacterial luminescence. The luminescence by purified luciferase preparations in the presence of FMNH2 and aldehyde was strongly inhibited by p-chloromercuribenzoate, a well-known sulfhydryl inhibitor. This was interpreted to mean that luciferase is a sulfhydryl enzyme. Inhibitors of luminescence such as cytochrome c, methemoglobin, ferricyanide, and menadione were found to be reduced by FMNH2 non-enzymically and in a quantitative manner and are believed to inhibit luminescence by competing for the hydrogen of FMNH2. The rate of FMNH2 oxidation by these inhibitors was found to be faster than the autoxidation rate of FMNH2 in air. Ferric and ferrous iron will inhibit luminescence but not nearly so well as the corresponding 8-hydroxyquinoline chelates. The evidence indicates that ferrous iron and its chelate inhibit by combining with luciferase, possibly through the sulfhydryl groups present. Hemin has been found to combine very effectively with luciferase and is one of the most potent inhibitors of bacterial luminescence known. It is not known whether the hemeprotein formed from hemin and luciferase has an enzymic property.