Quantitative Enzymic Conversion of Cocarboxylase (Vitamin B 2 -Pyrophosphate) to Free Thiamin

Abstract

Recently Lohmann and Schuster 1 reported that thiamin (vitamin B₁) may exist in nature as a pyrophosphoric acid ester to constitute cocarboxylase, the coenzyme of carboxylase. Thiamin itself cannot function as the coenzyme, although the action of pure cocarboxylase in vitro is strongly stimulated by the addition of the pure vitamin. 2 However, pigeon curative tests have shown that cocarboxylase is practically as equally effective as free thiamin. A number of workers 3 4 5 6 have subsequently reported that in the thiochrome method for determining thiamin the cocarboxylase forms a thiochrome but this is not extracted by isobutanol and hence cannot be estimated. 4 For this reason analyses by this method of preparations rich in cocarboxylase (such as yeast), give values decidedly less than those obtained by biological assay. 6 Cocarboxylase may be hydrolyzed 1 to the monophosphoric acid ester of thiamin by 1 N HCl solution, but the latter ester is resistant to acid hydrolysis. Alkaline hydrolysis will completely remove the second phosphoric acid grouping but inasmuch as there is a concomitant destruction of the end-product (thiamin) such a procedure is impractical for analytical purposes. Levine 6 has observed that, when aqueous yeast extracts are allowed to stand at room temperature exposed to the atmosphere for a period of 48 hours with no preservative added, there is a conversion of the cocarboxylase present to free thiamin. He attributed this change to the activity of a yeast phosphatase. Kidney phosphatase 1 5 7 has been used as means for hydrolyzing the coenzyme to the free vitamin. However, with such a preparation the conversion does not appear to be quantitative.

In the present study an extension of our chemical method 8 for the estimation of thiamin in biological materials has been applied to an investigation of the conversion of cocarboxylase to thiamin. We have found that both cocarboxylase and the monophosphoric acid ester of thiamin† react with diazotized p-amino acetophenone to yield colored solutions but that these pigments are not extracted by xylene. This is comparable to the findings recorded with the thiochrome method for determining thiamin.