DETERMINATION OF COCARBOXYLASE WITH ETIOZYMASE

Abstract

1. Using commercial dry yeast, Goodhart's method was reinvestigated, and a revised manometric method for the estimation of cocarboxylase was described.
2. Main revises were as follows: The amount of the thiamine added, the phosphate buffer concentration, and the substrate concentration were increased, whereas the concentration of etiozymase was decreased.
3. The improved method was found to be far more sensitive than the original method. The amount of 0.04 to 1.0γ of cocarboxylase could be determined accurately.
4. This method is not always applicable directly to tissue extracts, especially when glucose is present in the sample. But, for investigating the synthesis and degradation of cocarboxylase in the purified enzyme systems, this method will be useful and have sufficient sensitivity and accuracy.
5. The mechanism of the increase in sensitivity was studied.
It was proved more directly that thiamine prevented the decomposition of cocarboxylase.
6. The phosphate buffer concentration is a very important factor. When the phosphate concentration was increased, and the osmotic pressure reached nearly isotonic, the static denaturation of etiozymase was prevented. However, the osmotic factor was only a part of the effect of phosphate.
7. As another function of phosphate it was assumed that phosphate retarded the dynamic denaturation which took place only when etiozymase was actively catalyzing the reaction.