COMPETITIVE ANALOGUE-METABOLITE INHIBITION 473 



functions in the biosynthesis of these substances. 51 The inhibition index 

 obtained with methylfolic acid, a competitive analogue of folic acid, is 

 30 in the absence of purines, 100 in their presence, and 1000 in the pres- 

 ence of both purines and thymine. 51 Thymine is inactive alone. For Strep- 

 tococcus faecalis R, the toxicity of methylfolic acid is completely pre- 

 vented by both purines and thymine. 52 Purines alone exert a very slight 

 effect on the inhibition index, and thymine alone exerts no effect. An 

 involvement of folic acid in the biosynthesis of serine in Streptococcus 

 faecalis R 53 appears to be similar to the involvement of p-aminobenzoic 

 acid in the same biosynthesis in Escherichia coli. 



The involvement of p-aminobenzoic acid in biosyntheses involving a 

 single carbon unit led to a search for folic acid derivatives capable of 

 serving as a formate carrier. Pteroylhistidine which was prepared syn- 

 thetically did not exert any pronounced activity. However, the structure 

 of rhizopterin, N 10 -formylpteroic acid, gave a clue as to how formate 

 could be carried by a functional derivative of folic acid. Accordingly, 

 formylfolic acid was prepared and found to be approximately 30 times 

 as active as folic acid in preventing the toxicity of methylfolic acid for 

 Streptococcus faecalis R. The derivative was just as active as folic acid 

 in promoting growth of this and a number of other organisms requiring 

 folic acid. 54 



While it is not possible as yet on the basis of published results to con- 

 clude whether p-aminobenzoic acid and folic acid are converted to an 

 identical coenzyme, or whether two or more different coenzymes are in- 

 volved, inhibition analysis applied to natural extracts has been useful in 

 the discovery of a form of folic acid which is more widely active, and of 

 other related factors from which a solution to the perplexing problem 

 appears possible in the near future. 



Utilization of Inhibition Analysis in the Development of Assays for 

 Naturally Occurring Unknown Factors 



The techniques of inhibition analysis can be used to develop micro- 

 biological assays for unknown factors which may be difficult to detect by 

 other means. This offers a specific, direct approach to the discovery and 

 isolation of factors related to a specific metabolite. Such a problem pre- 

 sented itself recently when folic acid was found to be effective in the 

 treatment of pernicious anemia, but was not the anti-pernicious anemia 

 principle (s) of refined liver extracts. It was highly desirable at that time 

 to develop specific assays for unknown substances related to folic acid 

 or p-aminobenzoic acid and occurring in refined liver extracts. It has been 

 reported that through the use of inhibition analysis and related ap- 

 proaches more than twenty different assays have been developed for 



