Abadeh S, Killacky J, Benboubetra M, Harrison R
Biochemistry Department, University of Bath, UK.
Biochim Biophys Acta. 1992 Jul 21;1117(1):25-32. doi: 10.1016/0304-4165(92)90157-p.
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.
黄嘌呤氧化酶是从人乳中纯化得到的，其产量与从牛乳中获得的产量相当。新纯化的酶在凝胶渗透快速蛋白质液相色谱（FPLC）和十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）中呈现均一性，这与其作为总分子量为290,000±6000的同型二聚体一致。紫外/可见吸收光谱与牛乳酶的吸收光谱仅有轻微差异，其A280/A450比值为5.13±0.29，表明纯度很高。纯化酶的黄嘌呤氧化酶活性因乳的批次而异，范围在3至46 mU/mg蛋白质之间；这些值比牛乳酶的最高纯化样品所显示的值小约两到三个数量级。与市售牛乳酶的直接比较表明，以黄嘌呤作为还原底物的活性是牛酶活性的1 - 6%，而涉及还原型辅酶Ⅰ（NADH）的活性，相比之下，两种酶的活性处于同一水平。厌氧漂白实验表明，人酶中以对黄嘌呤有活性的形式存在的不到2%。这些发现与活性数据一起表明，人酶中脱钼和/或脱硫形式的含量非常高，可能大于98%，这两种形式在牛乳黄嘌呤氧化酶中也有少量存在。钼含量测定表明，脱钼酶仅占这种无活性成分的约26%，这表明脱硫酶可能占其余部分。
