Bovine milk xanthine oxidase: purification by ultrafiltration and conventional methods which omit addition of proteases: some criteria for homogeneity of native xanthine oxidase.
Methodological difficulties have been encountered when proteases were omitted from the conventional isolation of bovine milk xanthine oxidase (xanthine:oxygen oxidoreductase, EC 1.2.3.2). The use of these conventional methods has been studied and modified to reduce the problems encountered. Some of the difficulties may be due to the presence of high concentrations of caseins, which exhibit a wide range of charges and sizes, thereby making separations based on charge and size more complicated. In addition, non-covalent interactions may occur between the caseins and xanthine oxidase leading to the formation of casein-xanthine oxidase micellar aggregates. The difficulties encountered in this conventional isolation have been circumvented by purifying the enzyme directly from milk fat globule membranes that first have been washed free of most casein and other milk proteins. The xanthine oxidase is isolated by ultrafiltration through an Amicon XM-100A membrane at 5 degrees C in 0.25 M sucrose/5 mM sodium salicylate. The largest molecular size of globular proteins which can penetrate this ultrafiltration membrane has been previously estimated to be around 100 000 daltons. Xanthine oxidase thus appears to be smaller than 100 000 daltons in its native state. The size observed for active xanthine oxidase previously isolated by other methods has been around 275 000--300 000 daltons. Xanthine oxidase isolated by ultrafiltration appears similar to xanthine oxidase from conventional isolation methods according to empirical criteria of homogeneity based on size and also on the absorbances at 280 and 450 nm. Criteria based on charge were found to be less reliable.
D000645:Ammonium Sulfate; D000818:Animals; D002417:Cattle; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D005260:Female; D008892:Milk / Q000201:enzymology*; D008970:Molecular Weight; D010447:Peptide Hydrolases; D014462:Ultrafiltration; D014969:Xanthine Oxidase / Q000302:isolation & purification*
D014969:Xanthine Oxidase; D010447:Peptide Hydrolases; D000645:Ammonium Sulfate