High-performance affinity chromatography of NADP+ dehydrogenases from cell-free extracts using a nucleotide analogue as general ligand.
An epoxy-activated silica column (50 cm x 0.45 cm I.D.) was derivatized with 8-[6-aminohexyl)amino]-2'-phosphoadenosine-5'-diphosphoribose; the bound ligand concentration was 11.4 mumol/g of dry silica, and the useful loading capacity was 2.3 mg of glutathione reductase. The new high-performance liquid chromatographic column specifically retained NADP(+)-dependent enzymes, which were quantitatively eluted specifically by NADP+ or, with better resolution, by potassium chloride. The new high-performance liquid chromatographic support was applied to the purification of glutathione reductase and glucose-6-phosphate dehydrogenase from cell-free extracts of baker's yeast, fish liver and rabbit hemolysates, with high recoveries and excellent purification factors.
Alhama
J
J
Departamento de Bioquímica y Biología Molecular, Facultad de Veterinaria, Universidad de Córdoba, Spain.
López-Barea
J
J
Toribio
F
F
D000818:Animals; D002474:Cell-Free System; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005399:Fishes; D005954:Glucosephosphate Dehydrogenase / Q000097:blood / Q000302:isolation & purification; D005980:Glutathione Reductase / Q000097:blood / Q000302:isolation & purification; D008099:Liver / Q000201:enzymology; D009249:NADP / Q000494:pharmacology*; D010088:Oxidoreductases / Q000302:isolation & purification*; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000201:enzymology
D009249:NADP; D010088:Oxidoreductases; D005954:Glucosephosphate Dehydrogenase; D005980:Glutathione Reductase