III. BIOCHEMICAL SYSTEM 601 



fraction of a per cent. This was, however, enough to make Dr. Guirard 

 suspect that pantothenic acid might be present but might not be Hberated 

 effectively by the procedure used. She therefore tested for the presence of 

 ;S-alanine in the acid hydrolyzate of the coenzyme preparations; indeed, 

 the yeast assay showed /3-alanine to be present in amounts corresponding 

 to a pantothenic acid content of 11 %}'^' ^^ In view of the rare occurrence of 

 jS-alanine, it was then considered almost certain that pantothenic acid was 

 part of CoA. 



Because of the instability of pantothenic acid to alkali or acid, chemical 

 hydrolysis of CoA could not be applied for its liberation. Eventually the 

 unhinging of pantothenic acid from its links in CoA was accomplished by 

 enzymatic hydrolysis with intestinal phosphatase in conjunction with a 

 particular peptidase present in bird liver,^* in pig kidney,^* and, to a lesser 

 extent, in other organs. 



2. Relation of the Enzymatically Determined CoA Unit 

 TO Its Pantothenic Acid Content 



A unit of CoA corresponds to 0.7 7 of pantothenic acid per milligram. ^^^ ^' 

 This ratio has been of great value for assessment of purity; 1 )uM. of CoA 

 containing 1 fjM. of pantothenic acid corresponds to 310 units of CoA. 

 The molecular weight of the coenzyme was earlier determined by the 

 Northrup diffusion method^^ to be around 800. Therefore, pure CoA was 

 expected to contain approximately 400 units per milligram. Analytical 

 data obtained with our purest preparations,^^ which will be discussed below 

 in detail, indicate a molecular weight of 767 and a content of 410 units 

 per milligram of pure CoA, in excellent agreement with earlier predictions. 



3. Arsenolysis Assay 



During the work on isolation, we became aware that the assay system 

 with crude pigeon liver extract is not specific for intact CoA, but responds 

 to certain fragments.'^ This is due to enzymatic resynthesis of CoA from 

 partially dephosphorylated coenzyme in the presence of ATP. Products 

 which may be resynthesized to CoA have been obtained by controlled 



12 F. Lipmann, N. O. Kaplan, G. D. Novelli, L. C. Tuttle, and B. M. Guirard, J. 

 Biol. Chem. 167, 869 (1947). 



13 F. Lipmann, N. O. Kaplan, G. D. Novelli, L. C. Tuttle, and B. M. Guirard, /. 

 Biol. Chem. 186, 235 (1950). 



" G. D. Novelli, N. 0. Kaplan, and F. Lipmann, /. Biol. Chem. 177, 97 (1949). 



1^ L. Levintow and G. D. Novelli, Ahstr. Papers, Meeting Am. Chem. Soc. Atlantic 



City, p. 33C (Sept. 14-19, 1952). 

 16 G. D. Novelli, R. M. Flynn, and F. Lipmann, J. Biol. Chem. 177, 493 (1949). 

 " J. D. Gregorj', G. D. Novelli, and F. Lipmann, /. Am. Chem. Soc. 74, 854 (1952). 

 18 G. D. Novelli, N. O. Kaplan, and F. Lipmann, Federation Proc. 9, 209 (1950). 



