Abstract
Xanthine oxidase was purified from human milk in yields comparable with those obtained from bovine milk. The freshly purified enzyme appeared homogenous in gel permeation FPLC and SDS-PAGE, consistent with its being a homodimer with total M r290 000 ± 6000. The ultraviolet/visible absorption spectrum differed only by slightly from that of bovine milk enzyme and showed an A 280/ A 450 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 were 1–6% that of the bovine enzyme, whereas those involving NADH, in contrast, were of the same order for the two enzymes. Anerobic 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 indicated component, suggesting that desulpho-enzyme may account for the remainder.
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More From: Biochimica et Biophysica Acta (BBA) - General Subjects
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