title: Purification and partial characterization of xanthine oxidase from human milk.
abstract: Xanthine oxidase was purified from human milk in yields comparable with those obtained from bovine milk. The freshly purified enzyme appeared homogeneous in gel permeation FPLC and SDS-PAGE, consistent with its being a homodimer with total M(r) 290,000 +/- 6000. The ultraviolet/visible absorption spectrum differed only slightly from that of bovine milk enzyme and showed an A280/A450 ratio of 5.13 +/- 0.29, indicating a high degree of purity. Xanthine oxidase activities of purified enzyme varied with batches of milk, ranging between 3 and 46 mU/mg protein; values that are some two to three orders of magnitude smaller than those shown by the most highly purified samples of bovine milk enzyme. Direct comparison with commercially-available bovine milk enzyme showed that activities involving xanthine as reducing substrate were 1-6% that of the bovine enzyme, whereas those involving NADH, in contrast, were of the same order for the two enzymes. Anaerobic bleaching experiments indicated that less than 2% of the human enzyme was present as a form active with xanthine. These findings, together with the activity data, are consistent with a very high content, possibly greater than 98%, of demolybdo- and/or desulpho-forms of human enzyme, both of which occur, to a lesser extent, in bovine xanthine oxidase. Molybdenum assay indicated that demolybdo-enzyme could only account for some 26% of this inactive component, suggesting that desulpho-enzyme may account for the remainder.
authors.affiliation: Biochemistry Department, University of Bath, UK.
authors.forename: S
authors.initials: S
authors.lastname: Abadeh
authors.forename: J
authors.initials: J
authors.lastname: Killacky
authors.forename: M
authors.initials: M
authors.lastname: Benboubetra
authors.forename: R
authors.initials: R
authors.lastname: Harrison
mesh_terms: D000818:Animals; D000906:Antibodies / Q000032:analysis; D006801:Humans; D008892:Milk / Q000201:enzymology; D008895:Milk, Human / Q000201:enzymology*; D008970:Molecular Weight; D015427:Reperfusion Injury / Q000201:enzymology; D013053:Spectrophotometry; D014969:Xanthine Oxidase / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D014976:Xanthopterin / Q000032:analysis
chemical_list: D000906:Antibodies; C008217:isoxanthopterin; D014969:Xanthine Oxidase; D014976:Xanthopterin