A glycerol dehydrogenase from Escherichia coli.

In the course of studies of t.he reduction of Furacin (5-nitro-2-furaldehyde semicarbazone) by cell-free extracts of a strain of Escherichia co& the presence of a number of diphosphopyridine nucleotide-linked dehydrogenases was indicated by the ability of such extract’s when dialyzed to reduce the nitrofuran only on the addition of diphosphopyridine nucleotide (DPN) and any one of a number of oxidizable substrates.1 Reduction of the nitrofuran apparently was effected by an enzyme, presumably the flavoprotein described by Brodie (2), capable of mediating the transfer of electrons from reduced DPN (DPNH) to the nitrofuran. Attempts to demonst,rate spectrophotometrically reduction of DPN+ by these specific substrate-dehydrogenase systems then were unsuccessful. This inability to detect substrate-dehydrogenase reduction of DPN+ appears to be attributable to the presence of the flavoprotein previously mentioned (2), which could be shown to be capable of reoxidizing DPNH, with oxygen as the ultimate electron acceptor, at a sufficiently rapid rate to prevent its spectrophotometric detection in such experiments. However, one of these dehydrogenases, glycerol dehydrogenase, proved sufficiently heat-stable to be quite easily isolable in a highly active state, by the simple device of subjecting the crude cell-free extract to sufficient heat to dest.roy most, if not all, of the other oxidative enzymes present. After some additional purification by precipitation with ammonium sulfate, and subsequent dialysis, the physical and biochemical properties of this dehydrogenase were further studied. Evidence is here presented that oxidation of glycerol in the presence of this enzyme requires no phosphate, is specifically DPN-linked, and results in the production of dihydroxyacetone in a 1: 1 molar ratio with the DPN+ reduced.