600 



PANTOTHENIC ACID 



50 r 



12 3 



Units coenzyme A 



Fig. 2. Coenzj'me A assaj\ Concentration-activity curves for coenzyme A prepa- 

 rations of different purity. The arrow indicates the half-reactivation point which 

 represents our unit. O = crude coenzyme, 0.25 unit per milligram; X = purified 

 coenzyme, 130 units per milligram. 



straight line. A modified assay procedure with the same enzyme solution 

 using the color change caused by acetylation of 4-aminoazobenzene was 

 recently described by Handschumacher et al}^ This color change may be 

 measured directly. On the same principle, Bessman^^" in our laboratory re- 

 cently worked out a more convenient procedure using the much more solu- 

 ble 4-aminoazobenzene sulfonate. 



In view of the likelihood that a new coenzyme may contain one of the 

 vitamins not covered then by coenzyme activity, the preparation was 

 analyzed for its vitamin content. Fortunately, in this task we were helped 

 by the experience of Dr. Roger Williams' laboratory with vitamin assay 

 in general, and in particular with pantothenic acid assay. Dr. Beverly 

 Guirard, who carried out the analysis of our coenzyme preparations, found 

 little or no activity with Lactobacillus arahinosus. However, after incubation 

 for a prolonged period with clarase and papain — at that time considered 

 enzymes which liberated the "protein-bound" pantothenic acid of natural 

 sources — a small amount started to appear, still only of the order of a 



'1 R. E. Handschumacher, G. C. Mueller, and F. M. Strong, /. Biol. Chem. 189, 335 



(1951). 

 "" S. P. Bessman and F. Lipmann, Arch. Biochem. and BiopMjs. 46, 252 (1953); 



cf . also M. E. Jones, S. Black, R. M. Flynn, and F. Lipmann, Biochim. et Biophys. 



Acta 12, 143 (1953). 



