350 RIBOFLAVIN 



xanthine oxidase and the xantliopterin oxidase of cream are identical. 

 2-Aniino-4-hy(h'i)iitoridino is also oxidized by xanthine oxidaseJ^ 



!). Liver Aldiohydk Oxidase 



AVhile alteniptinij; (o prepare (he xanthini^ oxidase of pijz; li\er, Ciordon 

 ct al.''' isolaUnl a (hu'oprotein which sp(>ci(ically calalyzed Ihe oxidation of 

 aldehydes to their corresponding acids. 'The prosthetic j2;ronp was flavin 

 adenine diinicleotide. Unlike its connterpart in milk, this a Idehydase showed 

 no activity toward either hypoxanthine or reilnced coenzyme I. 



a. Preparation 



The removal of fonr main colored inipin'itics hemoglobin, xanthine oxi- 

 dase flavoprotein, a Fe(OH)3-protein complex, and catalase — -served as the 

 workinij; hypothesis npon which Gordon c( al." based their isolation. Hemo- 

 globin was separated by precipitating the aldehydase with half-saturatetl 

 ammoninm snlfatc. Xanthine oxidase was destroyed by heating at 48° for 

 5 minntes in 25% idcohol. The orange-rcxl Fe(()TT);rprotein complex (prob- 

 ably ferritin) is slightly less soluble than the aldehyde oxidase in ammonium 

 sulfate solutions. Repeated fractionations between 20 and 35 % saturations 

 of ammoniacal ammonium sulfate solutions removed all the iron complex. 

 The removal of catalase proved most dithcult, and, since some catalase 

 accompanies the aldehyde enzyme in the course of many salt fractionations, 

 adsorptions, solvent precipitations, cataphoresis, etc., it was believed that 

 catalase formed a compound with the aldehyde enzyme which could nt>t 

 be resolved by known methods. 



b. Propcrlics of Liver Aldehijde Oxidase 



Turilied [irc^parations of the enzyme are yelK)wish brown in color. This 

 color is partly bleached on reduction with hytlrosultite and restored after 

 shaking in air. It contains 0.17% flavin pht)sphate and has a turnover 

 nundier of about 550 with acetaldehyde. The flavin adenine dinucleotide 

 prosthetic group can he split off by boiling, acidification to less than pll 

 4, prolonged dialysis against water, or exj)osure to fat solvents, etc. 



Gordon et (?/." reasoned that their lixer flavoprotein was different from 

 the milk xanthine-aldehyde-dihydrocoenzyme I oxidase of Corran et a/.''-' 

 because the liver enzyme was somewhat less soluble in ammonium sulfate 

 solutions and was rapidly and irre\ersibly inactivated by dialysis against 

 water at 0°, wluMvas the n\ilk enzyme was stable o\cr a jicriod of days. 

 Furthermore, the cyclical reiluction and oxiilatioii of the flavin group in 



'^ O. 11. Lowry, O. A. Hossoy, aiul K. .1. Crawford, ./. Biol. Chcni. 180, ;iS!), :M)!) (1949). 

 7« J. N. Williams and C. A. Klvohjom, I'roc. Sov. Expll. Hiol. Mat. 71, Ml (1949). 

 " A. 11. ClDnloii, D. Iv Crc(Mi, ami V, Suhrahiuanyaii, Biochcni. ./. 34. TlVt (1940). 



