COENZYMES DERIVED FROM B VITAMINS 155 



boxylations. Also, there are instances in which the reactions requiring 

 "cocarboxylase" do not produce or utilize carbon dioxide. 



Thiamine pyrophosphate can be prepared synthetically by chemical 

 phosphorylation of the vitamin in and can be purchased from commercial 

 firms which stock biochemicals. 



The conversion of thiamine to the phosphoric acid ester profoundly 

 affects its biological reactivity even though, chemically, the change is not 

 a drastic one. Upon oxidation the coenzyme forms a fluorescent compound, 

 thiochrome pyrophosphate, by a reaction analogous to that which pro- 

 duces thiochrome from the vitamin;* the coenzyme can also be cleaved 

 by sulfurous acid into a pyrimidine and a phosphorylated thiazole; 110 

 the phosphorylation of the vitamin does not alter the susceptibility of 

 the molecule to cleavage by the "anti-thiamine" enzyme present in raw 

 fish for the coenzyme is split just as rapidly by this means as is the 

 vitamin. 



The most obvious chemical change in forming the coenzyme is the 

 creation of a strongly acidic compound from the organic base. This is 

 accompanied by an increase in the resistance of the thiazole nucleus to 

 reduction and reoxidation by chemical agents, 112 a fact of some impor- 

 tance when considering possible mechanisms of the functioning of thi- 

 amine (p. 168). 



Assay Methods. The determination of the thiamine pyrophosphate 

 content of natural materials is usually carried out by a manometric pro- 

 cedure in which the rate of pyruvic acid decarboxylation is followed. 

 The crude preparations of the apoenzyme needed for the procedure are 

 obtained by washing dried yeast cells with an alkaline phosphate buffer. 110 

 The extent of reactivation of the washed cells by extracts (in the presence 

 of Mg ++ ) is a direct measure of their coenzyme content. 



Thiamine itself often shows some activity when it is added to crude 

 apoenzyme preparations, presumably because of the presence of phos- 

 phorylating enzymes which convert the vitamin to the coenzyme during 

 the course of the determination. When brewers' yeast is used as a source 

 of the apoenzyme, free thiamine is found to exert an "activating" effect 

 upon the carboxylase enzyme. 113 Thiamine itself is completely inactive 

 when added to the apoenzyme, yet when added to the apoenzyme along 

 with the coenzyme, it increases the activity of the reconstructed system. 

 This effect is observed even when the coenzyme is present in excess ; hence 

 the thiamine effect cannot be attributed to a direct synthesis of additional 

 coenzyme. This "activation" effect is believed to be due to the ability of 



* The acidic pyrophosphoric acid ester of thiochrome (obtained from the coenzyme) 

 is not extracted from alkaline solutions by butyl alcohol; hence, the coenzyme does 

 not interfere in the thiochrome assay for the free vitamin when the usual procedures 

 are used (p. 46). 



