Abstract
This chapter describes the assay method, purification procedure, and properties of D-galactose-6-phosphate isomerase. The D-galactose-6-phosphate isomerase enzyme is instrumental in the catabolism of lactose and D-galactose in Staphylococcus aureus . The continuous spectrophotometric assay based on the sequence of reactions is also discussed in the chapter. With the excess of the three coupling enzymes formed during the reactions, the rate of D-galactose 6-phosphate isomerization is equivalent to the rate of nicotinamide adenine dinucleotide dehydrogenase (NADH) oxidation, which is measured by the absorbance decrease at 340 nm. The steps involved in the purification of the enzyme are (1) the preparation of cell extracts, (2) bentonite treatment, (3) negative adsorption on hydroxyapatite, (4) diethylaminoethyl (DEAE)-cellulose chromatography I and II, and (5) Sephadex G-100 chromatography. The enzyme in the Sephadex G-100 fractions is stable for several weeks during storage at –20°C. The rates are measured with a Gilford multiple-sample absorbance-recording spectrophotometer. The molecular weight of the enzyme is estimated to be about 99,000-100,000 by both sucrose gradient centrifugation and Sephadex chromatography.
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