FUNCTION 



O. Warburg and W. Christian ^ had shown that the " yellow enzyme ", 

 of which riboflavine was later shown to be the prosthetic group, was 

 an essential link in the metabolism of carbohydrates. The series of 

 transformations whereby oxygen is transferred from a substrate 

 undergoing dehydrogenation to atmospheric oxygen, is discussed in 

 the chapter on nicotinic acid (see page 277), where it is stated that the 

 yellow enzyme, diaphorase, oxidises reduced coenzyme (di- or tri- 

 phosphopyridine nucleotide) and is itself re-oxidised (with one excep- 

 tion) by cytochrome. 



Actually the picture is complicated by the fact that riboflavine is 

 a constituent of two different coenzymes, the second of which can 

 catalyse several important transformations. The first of these co- 

 enz5nTies, known as riboflavine mononucleotide, was isolated from 

 Warburg and Christian's yellow enzjone by H. Theorell,^ who purified 

 the enzyme from yeast by cataphoresis, and split it by dialysis into 

 the apoenzyme (the protein carrier) and the coenzyme. 



Weygand et al.^ improved the isolation of the enzyme by adsorption 

 on " ortho aluminium hydroxide y " and elution with ammonium 

 sulphate at pU 5-2, followed by precipitation at pR 6 by 70 % satura- 

 tion with ammonium sulphate and then dialysis.* 



The molecular weight of the enzyme was estimated to be 

 74,000.^' ^ 



The nature of the coenzyme was established by R. Kuhn and 

 H. Rudy,'' who showed that " cytoflav ", a golden-yellow pigment 

 isolated from heart -muscle by L Banga and A. Szent-Gyorgyi ^ four 

 years before, was riboflavine-5 '-phosphoric ester. They prepared this 

 compound synthetically by treating riboflavine with phosphorus oxy- 

 chloride, and showed that the product had the same activity as natural 

 cytoflav. Moreover, it had the same growth-promoting activity on 

 rats, mole for mole, as riboflavine. The flavine-phosphoric ester 

 prepared from yeast by Theorell had similar properties. H. Rudy ^ 

 prepared riboflavine-phosphoric ester from riboflavine by digestion 

 with intestinal phosphatase. An improved method of synthesis was 

 described by Kuhn et al.^^ ; 5'-trityl-riboflavine was acetylated, giving 

 2' : 3' : 4'-triacetyl-5'-trityl-riboflavine, and the trityl group was then 

 removed and the product phosphorylated and de-acetylated. R. Kuhn 

 and H. Rudy ^^ completed their work by preparing a " synthetic " 

 enzyme ; sodium riboflavine-5'-phosphate was adsorbed on the 

 colloidal carrier of the yellow enzyme, giving a non-fluorescent, 

 non-dialysable chromoprotein, which decolorised methylene blue 

 at the same rate as the natural enzyme. They suggested that 

 the protein was probably linked to the riboflavine-phosphoric ester 

 at the phosphoric acid radical and the 3-imido group as in the^ 

 formula : 



191 



