IV. BIOCHEMICAL SYSTEMS 347 



more active {'ompoiiiuls with ril)o(la\in phosphate than with fla\in adenine 

 (hnucleolide. 



The reaelion of li\er cytochrome c retiuctase witli oxygen is less lium 2 % 

 as fast as the reaction with cytochrome c. 



The turnover number of the Uver enzyme is 1140 (molecular wei<>;ht 

 08,000), as compared to a similar turnover number of 1300 (molecular 

 weight 78,000) for the yeast enzyme. 



8. X.vNTHiNE Oxidase, Aldehyde Oxidase, Xanthopterin Oxidase 



The enzymatic o.xidation of hypoxanthine and xanthine to uric acid in 

 the presence of tissue brei and oxygen was recognized by Spitzer^* in 1899. 

 Buriaii^^ named this enzyme xanthine oxidase. In 1902, Schardinger'^^ ob- 

 served that if formaldehyde and methylene blue were added to fresh milk 

 in the absence of oxj^gen the methylene blue was rapidly decolorized. These 

 two enzymatic processes remained unrelated until 1922, when Morgan et 

 al}' showed that milk was a rich source of xanthine oxidase. The work of 

 Dixon and Thurlow,'^* Booth, ^^ and Ball''''' ^' supported the suggestion that 

 the same enzyme is involved in both reactions. Corran and Green^- isolated 

 a flavoprotein from cow's milk w'hich showed no activity as a xanthine- 

 aldehyde oxidase but could catalyze the oxidation of reduced coenzyme I. 

 Then Corran et al.^^ prepared a milk flavoprotein which catalyzed the oxi- 

 dation of hypoxanthine, aldehydes, and reduced coenzyme I, and showed 

 that although all three activities w^ere associated with the same flavoprotein 

 they could be differentially inactivated. Although the Michaelis constant 

 (/v„,) and the kinetics of the three substrates are not identical, the addition 

 of aldehyde specifically inhibits the oxidation of purine and vice versa. 

 Either the same active group is concerned in the activation of both purines 

 and the aldehydes, or else there are two active groups in such close juxta- 

 position in the protein miolecule that activity at one interferes wdth activity 

 at the other.^'* 



a. Preparation 



Ball^' has prepared xanthine oxidase from unpasteurized cream, since 

 the enzyme was adsorbed on the fat globules. After the cream was shaken 

 with one volume of 0.2 M Na2HP04 at 38° for 2 hours, the fat was re- 

 moved b}' centrifugation at 0°. The enzyme solution was digested with 

 commercial lipase for 3^2 hours at 38°, clarified with 0.5 M calcium chlo- 

 ride, and centrifuged. The solution was 60% saturated with ammonium 

 sulfate, and after standing overnight at 0° the enzyme was centrifuged 

 down, dissolved in one-tenth the original volume of water, and further 

 purified b\^ precipitating with 33 % ammonium sulfate. 



punueu uy precipiiaung wiin ^6 vo ammonium sun 

 " W. Spitzer, Pflugers Arch. ges. Physiol. 76, 192 (1899). 



