VI. ESTIMATION 21 



PABA also may be dctormiiietl cheniically'*^ by a proceduiv in wliich dia- 

 zotized thiamine couples with PABA to yield a colored product that is 

 measured quantitatively in tlie spectrophotometer after extraction into 

 isoamyl alcohol. 



;;-l)imethylaminobenzaldehyde reacts with aromatic amines to give a 

 yellow color. This reaction has been suggested as a method for the deter- 

 mination of sulfonamitles and PABA.'^--' The compound lesponsible for the 

 color with sulfanilamide is p-dimethylaminobenzylidene-p-aminobenzene- 

 sulfonamide. Presumably the compound responsible for the color with 

 PABA is the following: 



CH3 



N— <k >— CH=N— 4 i>— COOH 



CH3 

 p-Dimethylaminobenzylidene-p-aminobenzoic acid 



Rf \'alues for o-, m-, and p-aminobenzoic acid by paper chromatography 

 in the system butanol saturated with 5 N NH4OH were found to be 0.38, 

 0.19, and 0.12, respectively, by Lederer.^- Rf values by paper chromatog- 

 raphy for PABA, PAHA (p-aminohippuric acid), and PASA (p-amino- 

 salicylic acid) in the system w-butanol-water have been published by Kele- 

 men ei alP These values may be of aid in the quantitative determination 

 of PABA and related compounds, particularly in urine. 



B. .MICROBIOLOGICAL METHODS 



Prior to the discovery of microorganisms with a growth factor require- 

 ment for PABA, a number of investigators had examined natural material 

 microbiologically for sulfonamide inhibitor.-*--^ A test for "sulfonamide 

 inhibitor" that depends upon the activity of natural material in reversing 

 the bacteriostatic activity of sulfapyridine for Escherichio coli was employed 



'« K. R. Kirch and O. iiorKoim, ,/. Biol. ('hem. 148, 445 (1943). 



'^ H. Tauher and S. Laufer, ./. Am. Chem. Soc. 63, 1488 (1941). 



i» .\. ]•:. .\. Werner, Lancet I, 18 (1939). 



"C. J. Morris, Biochem. J. 35, 952 (1941). 



2»A. T. Fuller, Lancet I, 760 (1942). 



2' W. y. LaRosa, Proc. Soc. Exptl. Biul. Med. 53, 98 (1943). 



" M. Lederer, Australian J. Sci. 11, 208 (1949). 



" E. Kelemen, B. Tanos, and D. Halmdgyi, Biochem. J. 47, 138 (1950). 



"J. S. Lockwood, J. Immunol. 35, 155 (1938). 



" T. C. Stamji, Lancet 237, 10 (1939). 



"C. M. MacLeod, ./. Kxptl. Med. 72, 217 (1940). 



" J. S. Lockwood and H. M. Lynch, ./. Am. Med. .l.s-.sor. 114. 935 (1910). 



2' H. X. Green, Bril. ./. Exptl. Pathol. 21, .38 (1940). 



"A. Fleming, ./. I'nth'd. Barleriid. 50, 09 (19)0). 



