Abstract
Publisher Summary D-Galactose dehydrogenase catalyzes the pyridine nucleotide-dependent dehydrogenation of β -D-galactopyranose at C-1 with the formation of nicotinamide adenine dinucleotide dehydrogenase (NADH) and D-galactono-l,5-1actone. Depending on the pH value, the immediately formed D-galactono-l,5-1actone rearranges intramolecularly to the corresponding 1,4-1actone or hydrolyzes to D-galactonate, or both. The assay is based on the spectrophotometric determination of NADH formed with D-galactose as substrate at high pH. This chapter discusses the assay method and properties of D-galactose dehydrogenase. The steps involved in the purification procedure are (1) the preparation of cell-free extract (2) ammonium-sulfate fractionation, (3) bulk separation on CM-Sephadex, (4) diethylaminoethyl (DEAE)-sephadex chromatography, and (5) hydroxyapatite chromatography. All operations are carried out at 0-4°C, unless stated otherwise. The purified enzyme with a specific activity of 849units/mg is homogeneous by ultracentrifugation, by disc gel electrophoresis, by sodium dodecyl sulfate gel electrophoresis, and by gel isoelectric focusing. D-Galactose dehydrogenase from P. fluorescens has a molecular weight of 64,000. The enzyme is composed of two identical subunits and has two binding sites.
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