348 



RIBOFLAVIN 



The preparation by Corran et al.^^ started with whole milk and included 

 precipitation in 13 % alcohol and adsorption on alumina as well as precipi- 

 tation with ammonium sulfate. Although their process was a bit more com- 

 plicated and gave a lower yield, the product obtained had a catalytic activ- 

 ity which was about 1000 times greater than that of milk. 



Xanthine oxidase can also be prepared from pig's liver. ^^ 



6. Properties of Xanthine Oxidase 



The xanthine oxidase prepared by Ball^' had an isoelectric point at about 

 pH 6.2. The addition of cyanide alone to xanthine oxidase caused an irre- 

 versible inhibition. If cyanide and substrate were added simultaneously, 

 no inhibition occurred. The mode of action of cyanide, first noted by 

 Szent-Gyorgyi^^ and later studied by Dixon and Keilin,*^ remains unex- 

 plained. This inhibition was utilized by Corran et al.^^ to eliminate the 

 xanthine-aldehyde without affecting the diaphorase activity. This diapho- 

 rase activity to reduced coenzyme I has been confirmed by Ball and Rams- 

 dell.«7 



The molecular weight of Ball's preparation was calculated as 74,000. It 

 was golden brown in color and did not fluoresce in ultraviolet light. The 

 flavin adenine dinucleotide prosthetic group could be split off from the 

 apoenzyme by dialyzing against running water for 2 weeks. 



c. Action of Xanthine- Aldehyde Flavoproteins 



Hypoxanthine and xanthine are oxidized to uric acid in the presence of 

 the oxidase: 



HN— C=0 



H 



(HO)HC C— N 



HN— C=0 



H 



0=C C— N 



/ 



CH 



+ 2H 



/ 



CH 



HN— C— N 



Hydrated hypoxanthine 

 HN— C=0 



H 



HN— C— N 



Xanthine 

 HN— C=0 



0=C C— N 



HN— C— N 

 H 



0=C C— N 



CH(OH) 



\ + 2H 



c=o 



Hydrated xanthine 



HN— C— N 

 Uric acid 



