牛奶黄嘌呤氧化酶的纯化及性质研究

本文建立从牛奶中分离纯化黄嘌呤氧化酶的工艺，该工艺采用正丁醇-硫酸铵分级提取、磷酸钙凝胶吸附、SephadexG-150层析纯化获得纯品．从磷酸钙凝胶上解吸到的XOD粗酶比活性达5．096I．U／mg－pr，提纯340倍，产率达74．3％．每单位粗酶的吸附剂用量以6mg为最佳．纯酶的最大紫外吸收在285nm处，酶活性测定的最适pH在8．60～10．15之间，最适温度在25℃～35℃间．1mol／LKCN、1．5％H2O2及甲醇对酶活力都有明显的抑制作用．经聚丙烯酰胺凝胶电泳显示两条蛋白带．经黄嘌呤-黄嘌呤氧化酶-Luminol（XOD－X－Luminol）发光体系检测结果表明20μl牛奶XOD（相当0．181．UXOD）的发光强度最大，其发光值受抑制50％的超氧化物歧化酶（SOD浓度（IC50）为1．14μg／ml（相当0．514USOD）．

Milk xanthine oxidase: purification and properties

In this study, a technique has been built up for the purification of milk xanthine oxidase (XOD) by using n-butyl alcohol- (NH4)2SO4 fractionation, calcium phosphate gel adsorption and sephadex G-150 chromatography. Xanthine oxidase was isolated in good yield and purity from milk by choosing 6 mg calcium phosphate gel per unit of the enzyme to adsorb it. It was found that the ultraviolet absorption peak of xanthine oxidase was at 285urn. As for enzyme assay, the optimal PH was between 8. 60 and 10. 15,and the optimal temperature was between 25℃ and 35℃. The activity of XOD could be restrained obviously by 1 mol/LKCl, 1.5 % H2O2 and methyl alcohol. Polyacrylamide gel electrophoresis showed XOD to have two protein bands.Xanthine oxidase-Xanthine-Luminol luminescence system showed there was a chemiluminescence peak at 20μl XOD. The liminescent intensity could be restrained obviously by SOD. The amount of SOD concentration for scavenging 50% O2 was found to be 1.14μg/ml (0. 514 unit SOD).