Studies on decarboxylant enzymes. XVII. Enzymatic evaluation of thiamine diphosphate

Abstract

To date, the most specific method for the determination of thiamine pyrophosphate (DPT), is the enzymatic assay based on the manometric measurement of the CO2 evolved by the en­ zymatic decarboxylation of pyruvic acid. The employment of CO2 evolution as specific assay method needs the use of highly purified apoenzyme preparations, hitherto not available for ac- curate and reproducidle results. The isolation in our Laboratory of an apoenzyme preparation of high purity and yield — 100 % recovery — suggested its use for DPT estimation. The method described in this paper appears convenient for this purpose. Standard curves — /d CO2 plotted against micrograms of DPT — are repported, showing the accurancy of the procedure for amounts ranging from apoenzyme saturation to 0.01 /xg per 1.5 mi.