The Liberation of Pantothenic Acid from Coenzyme

It was reported previously that coenzyme A contained pantothenic acid bound in such a manner as to make it unavailable in microbiological tests (1). A liberation of p;alanine on acid hydrolysis indicated early the presence of pantothenic acid, which was confirmed by chick assay (2). While the chick assay for pantothenic acid and the p-alanine assay by the yeast growth test yielded equivalent amounts of pantothenic acid, the microbiological assay for pantothenic acid with Lactobacillus arabinosus was practically negative with the intact coenzyme. The problem arose, therefore, how to liberate pantothenic acid from the coenzyme. Since pantothenic acid itself is very sensitive to any severe treatment with strong acid or alkali, the use of enzymatic methods to free the vitamin seemed most hopeful. It had been observed variously that the coenzyme activity was destroyed enzymatically. It appeared probable that such destruction may be due to the removal from pantothenic acid of attached groups which were essential for coenzyme activity, but, on the other hand, inhibitory to the utilization of the vitamin by intact microorganisms. A very rapid inactivation had been found with preparations of intestinal phosphatase (3) as well as with the pigeon liver extract used in acetylation experiments (4). Inactivation by intestinal phosphatase was accompanied by a complete liberation of organically bound phosphate. Inactivation by the pigeon liver extract, however, proved not to be due to a removal of phosphate but to a so far undetermined reaction. A combination of the two agents eventually proved necessary for a complete removal from pantothenic acid of all attached groups. After the incubationof coenzyme Awith intestinal phosphatase and liver extract, the microbiological assay of the coenzyme checked well with the results obtained by chick assay and by the /3-alanine test with acid-hydrolyzed coenzyme. Only little capacity to liberate pantothenic acid in coenzyme was found with clarase, mylase, and prostate phosphatase. Enzyme Preparations