ENZYMES ATTACKING NUCLEIC ACIDS 611 



the livers and particularly in the intestines of weanling rats and some other 

 mammals.227 228 According to Westerfeld and his collaborators,--^ xanthine 

 dehydrogenase preparations purified by Ball's method contained 0.03% 

 molybdenum. It is possible that the molybdenum-containing group repre- 

 sents the "second active group" of xanthine oxidase already suspected by 

 Ball.222 



The role of molybdenum as a functionally important component of 

 xanthine oxidase was demonstrated by Totter,--*=^ by Green and Beinert,-^'' 

 and by Mackler et alr^^'^ Totter et al. found that labeled molybdate in- 

 jected into a cow appeared in the xanthine oxidase isolated from the milk, 

 and that the proportion between the molybdenum and the flavine remained 

 constant at the value of 0.5. Green and Beinert arrived at the same pro- 

 portion in their analyses of highly purified preparations of the enzyme. 

 According to Mackler et al, molybdenum can be removed from the enzyme 

 by dialysis in alkaline solution. The molybdenum-free enzyme catalyzes the 

 oxidation of substrate by indophenol or oxygen, but is incapable of reacting 

 with one-electron acceptors such as cytochrome c or ferric cyanide. On 

 addition of molybdic oxide, this catalytic function is restored. This specific 

 role of the metal in facilitating the reaction of the enzyme with one-electron 

 acceptors applies to other enzymes belonging to the group of metalloflavo- 

 proteins. 



The metal-free xanthine oxidase can be partially reactivated by uranyl 

 ions, but not by tungstate or by any representative of a large number of 

 other metals. The metal-catalyzed oxidation of the substrate by one-elec- 

 tron acceptors has an absolute requirement for phosphate ions. 



The role of molybdenum is not an exceptional property of xanthine 

 oxidase since the nitrate reductase of Neurospora crassa was shown by 

 Nason and Evans-''''= to contain flavine adenine dinucleotide and molyb- 

 denum as essential constituents. 



According to Corran et al.,'^-^ the xanthine oxidase of milk is most likely 

 identical with xanthine oxidase of liver, but some observations suggest that 

 the enzymes in milk and in tissues might be associated with different 

 carrier proteins. 



Assay Methods. Xanthine oxidase may be assayed by the reduction of 

 methylene blue,-'-^ •-^'' by the manometric determination of oxygen up- 



"8 D. A. Richeit and W. W. Westerfeld, J. Biol. Chevi. 203, 915 (1953). 



229 W. W. Westerfeld, J. M. McKibbiiis, J. C. Roemel, and M. F. Hilfinger, Avi. J. 

 Physiol. 157, 18-1 (1949). 



229a J. R. Totter, W. T. Burnett, Jr., R. A. Monroe, I. B. Witney, and C. L. Comer, 



Science 118, 555 (1953). 

 229b D. E. Green and H. Beinert, Biochim. et Biophys. Acta 11, 599 (1953). 

 2290 A. Nason and H. J. Evans, J. Biol. Chem. 202, 655 (1953). 



230 V. H. Booth, Biochem. J. 32, 494 (1938). 



