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 .
The problem of homogeneity of crystalline molybdenoflavoprotein fractions with high xanthine oxidase activity has been studied by sedimentation experiments, by electrophoresis, and by " constant protein " and " constant solvent " solubility. analysis. All these tests, although in some instances not producing complete information, indicated that in our best preparations a t least 90% of the material acted physicochemically as a single component. However, enzymic activity measurements, more fully discussed in Part I11 (following paper), made it clear that " active " and closely related " inactive " flavoproteins were present in this " component." From the sedimentation, diffusion, and density data, it has been calculated that the crystalline material has M about 290,000. Its isoelectric point is a t pH 5'3-5.4. AVIS et aZ.l recently described a procedure for the preparation from cow's milk of a molybdenoflavoprotein with high xanthine oxidase activity and a low protein-flavin ratio, obtained for the first time in a crystalline form. It appears that xanthine oxidase, like
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