Xanthine oxidase and aldehyde oxidase: a simple procedure for the simultaneous purification from rat liver.
Aldehyde oxidase (AO) and xanthine oxidase (XO) are cytosolic enzymes that have been involved in some pathological conditions and play an important role in the biotransformation of drugs and xenobiotics. The increasing interest in these enzymes demands for a simple and rapid procedure for their purification. This paper describes for the first time a method that allows simultaneous purification of both enzymes from the same batch of rat livers. It involves few steps, is reproducible and offers high enzyme yields with high specific activities. The rat liver homogenate was fractionated by heat denaturation and by ammonium sulphate precipitation to give a crude extract containing both enzymes. This extract was chromatographed on an Hydroxyapatite column that completely separated AO from XO. Further purification of XO by anion exchange chromatography on a Q-Sepharose Fast Flow column resulted in a highly purified (1200-fold) preparation, with a specific activity of 3.64 U/mg and with a 20% yield. AO was purified about 1000-fold at a yield of 15%, with a specific activity of 3.48 U/mg, by affinity chromatography on Benzamidine-Sepharose 6B. The purified enzymes gave single bands of approximately 300 kDa on a polyacrylamide gel gradient electrophoresis and displayed the characteristic absorption spectra of highly purified enzymes.
Maia
Luisa
L
Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, Edifício C8, Campo Grande, 1749-016 Lisboa, Portugal.
Mira
Lurdes
L
D042931:Aldehyde Oxidase; D000445:Aldehyde Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D000645:Ammonium Sulfate / Q000494:pharmacology; D000818:Animals; D001671:Biochemistry / Q000379:methods*; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D008099:Liver / Q000201:enzymology*; D008297:Male; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors; D014968:Xanthine Dehydrogenase / Q000737:chemistry / Q000378:metabolism; D014969:Xanthine Oxidase / Q000737:chemistry* / Q000378:metabolism
D014968:Xanthine Dehydrogenase; D014969:Xanthine Oxidase; D000445:Aldehyde Oxidoreductases; D042931:Aldehyde Oxidase; D000645:Ammonium Sulfate