Purification and partial characterization of xanthine oxidase from human milk.
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.
Abadeh
S
S
Biochemistry Department, University of Bath, UK.
Killacky
J
J
Benboubetra
M
M
Harrison
R
R
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
D000906:Antibodies; C008217:isoxanthopterin; D014969:Xanthine Oxidase; D014976:Xanthopterin