Kihachiro Uehara

[39] d-erythrulose reductase from beef liver

Publisher Summary This chapter describes the assay method of D-erythrulose reductase isolated from the beef liver. D-erythrulose reductase activity is measured by following the decrease in the absorption of nicotinamide adenine dinucleotide phosphate dehydrogenase [NAD(P)H] at 340 nm, accompanying the reduction of D-erythrulose. The reaction is initiated at 28° by the addition of enzyme. A blank without D-erythrulose must be run to correct for any nonspecific oxidation of NAD(P)H. The steps involved in the purification procedure are (1) homogenization, (2) acetone fractionation, (3) diethylaminoethyl (DEAE)-cellulose chromatography I and II, (4) hydroxyapatite chromatography I and II, and (5) crystallization. D-erythrulose reductase contains 2-3 mol of bound NADP+ per mole of enzyme, so that the extinction coefficient of the enzyme at 280 nm is not constant. Thus, the concentration of pure enzyme protein is calculated from absorbance measurement at 290 nm; at this wavelength, the contribution of NADP+ to the absorbance is negligible. The molecular weight of the enzyme is estimated by sedimentation equilibrium analysis, Sephadex Go200 gel filtration, and sucrose density gradient centrifugation. The subunit molecular weight is determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

[44] Preparation and analysis of dihydroxymaleate and hydroxypyruvate

Publisher Summary Dihydroxymaleic acid can be prepared by oxidation of tartaric acid by which crystalline dihydroxymaleic acid hydrate is collected. If recrystallization is necessary, 20 g. of the product is dissolved in 160 ml. of absolute alcohol at room temperature and the solution is filtered. The filtrate is cooled in an ice bath and gently stirred during the dropwise addition of 150 ml. of water; the crystalline dihydroxymaleie acid is collected; washed, and dried. The quantitative determination of dihydroxymaleic acid is based on the reduction property of it, in the presence of salts. The titrimetric method for the quantitative estimation of dihydroxymaleic acid consists in decomposing dihydroxymaleic acid by boiling the aqueous solution and determining the decrease in acidity by titration to phenolphthalein. The use of 2,6-dichlorophenolindophenol to determine dihydroxy- maleic acid is based on the fact that the colored indicator is quantitatively and rapidly reduced by dihydroxymaleic acid in acid solution to a colorless compound.