24 P-AMINOBENZOIC ACID 



amide, that PABA reverses sulfanilamide inhibition of both strains, and 

 that the presence of benzoic acid, p-hydroxybenzoic acid, or tyrosine does 

 not increase the amount of PABiV synthesized by the parent strain. 



In the development of an assay method for PABA with the Neurospora 

 crassa mutant it was observed by Thompson et al.^^ ■ ^° that water extraction, 

 autolysis, or enzymatic digestion of natural materials is not always sufficient 

 to release all the PABA contained in the sample. Autoclaving at 120° for 1 

 hour with 6 N H2SO4 was the procedure finally recommended for release 

 of "bound" PABA. 



As originally described, the Neurospora crassa method involves the prepa- 

 ration of a number of petri plates, each containing basal medium supple- 

 mented with a definite amount of PABA solution or material to be assayed. 

 An inoculum block is then placed on each plate and, following a 20-hour 

 period of growth, the diameter of the mold growth surrounding the inoculum 

 block is measured with calipers and is dependent on the amount of PABA 

 in the culture plate. From a response curve obtained with PABA the po- 

 tency of unknown samples may be calculated. Although the authors claim 

 a number of advantages for this method of assay, including rapidity and 

 freedom from contamination due to the short incubation period employed, 

 it would appear that the method is subject to a number of possible sources 

 of error owing to the cumbersome method of inoculating the test plates. 



Agarwala and Peterson^^ have described procedures for the determina- 

 tion of PABA with the PABA-less mutant of Neurospora crassa in which 

 the mold is grown in licjuid culture. Following growth of the organism in 

 response to PABA or unknown material, the mycelial pads are removed 

 from the flasks in which they grew and are dried and weighed in the 

 conventional manner. A variety of compounds (see Table IV) related 

 to PABA in structure, including folic acid, were found to be devoid of 

 microbiological activity. Acid and alkaline hydrolysis were studied as 

 methods for the liberation of "bound PABA." Although either method of 

 hydrolysis gives higher apparent PABA values than are obtained without 

 hydrolysis, the authors point out that the increments observed probably 

 represent destruction of folic acid. Since the structure of folic acid was 

 unknown at the time that most of the microbiological methods for the de- 

 termination of PABA were worked out, many of the data concerning the 

 distribution of "bound PABA" (see Table V, p. 30) obtained by micro- 

 biological methods of assay are subject to re-evaluation. 



The essential nature of PABA (along with biotin) for Closindium aceto- 

 butylicum forms the basis for the microbiological methods for the deter- 

 mination of PABA with this organism.'*''-''" 



" S. C. Agarwala and W. H. I'etersoii, Arch. Hiochcm. 27, 304 (1950). 

 "^ S. D. Hubbo, M. E. Maxwell, R. A. Fairbridgo, and J. M. Cfillospic, Australian J. 

 Exptl. Biol. Med. Sci. IS, 185 (1941). 



