The composition of milk xanthine oxidase has been reinvestigated. When the enzyme is prepared by methods that include a selective denaturation step in the presence of sodium salicylate the product is obtained very conveniently and in high yield, and is homogeneous in the ultracentrifuge and in recycling gel filtration. It has specific activity higher than previously reported preparations of the enzyme and its composition approximates closely to 2mol of FAD, 2g-atoms of Mo and 8g-atoms of Fe/mol of protein (molecular weight about 275000). In contrast, when purely conventional preparative methods are used the product is also homogeneous by the above criteria but has a lower specific activity and is generally comparable to the crystallized enzyme described previously. Such samples also contain 2mol of FAD/mol of protein but they have lower contents of Mo (e.g. 1.2g-atom/mol). Amino acid compositions for the two types of preparation are indistinguishable. These results confirm the previous conclusion that conventional methods give mixtures of xanthine oxidase with an inactive modification of the enzyme now termed ;de-molybdo-xanthine oxidase', and show that salicylate can selectively denature the latter. The origin of de-molybdo-xanthine oxidase was investigated. FAD/Mo ratios show that it is present not only in enzyme purified by conventional methods but also in ;milk microsomes' (Bailie & Morton, 1958) and in enzyme samples prepared without proteolytic digestion. We conclude that it is secreted by cows together with the active enzyme and we discuss its occurrence in the preparations of other workers. Studies on the milks of individual cows show that nutritional rather than genetic factors determine the relative amounts of xanthine oxidase and de-molybdo-xanthine oxidase. A second inactive modification of the enzyme, now termed ;inactivated xanthine oxidase', causes variability in activity relative to E(450) or to Mo content and formation of it decreases these ratios during storage of enzyme samples including samples free from demolybdo-xanthine oxidase. We conclude that even the best purified xanthine oxidase samples described here and by other workers are contaminated by significant amounts of the inactivated form. This may complicate the interpretation of changes in the enzyme taking place during the slow phase of reduction by substrates. Attempts to remove iron from the enzyme by published methods were not successful.

A procedure for the preparation of a crystalline metallo-flavoproteh from buttermilk is described. It possesses high xanthine oxidase activity (QO, = 2300; spectrophotometric assay with xanthine at 23.5") and a protein/flavh ratio (ie., EZm/E4=) of 54-5-2, the lowest reported for material with such enzymic properties. 210, 149). Their products were not crystalline and none of the authors claimed a homogeneous product. The identity, or otherwise, of the bovine milk enzyme with the xanthine oxidases of animal and human somatic tissues has not so far been ascertained.Using improved preparative methods we have obtained from buttermilk an active crystalline metallo-flavoprotein as reported in a preliminary communication (Avis, Bergel, Bray, and Shooter, Nature, 1954, 173, 1230). In the present paper we describe our preparative procedures in detail and give the relevant assay figures.The progressive purification of the enzyme was followed by means of the following measurements : optical densities at 280 mp (" protein ") and 450 mp (" flavin ") (cf. Corran et al., b c . cit.), and the slope AEm5 %/At(-.> in the presence of xanthine (" enzyme activity ") (Kalckar, J . Biol. Chem., 1947, 167, 429; Morell, Zoc. n't.). Details of these assay methods and definitions of the unit of activity are given in the Experimental section, where also evidence can be found that solutions of the enzyme, unlike certain other proteins (cf. Champagne, J .

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