^-AMINOBENZOIC ACID 



2. ISOLATION OF p-AMINOBENZOIC ACID 



As already mentioned (page 546), the isolation of ^-aminobenzoic 

 acid from yeast and its unequivocal identification was first accom- 

 plished by S. D. Rubbo and J. M. Gillespie/ but an improved method 

 of isolation was described by K. C. Blanchard ^ in the following 

 year. 



An aqueous alcoholic extract of yeast was extracted with ether, 

 and the ethereal extract evaporated. The residue was made slightly 

 alkaline with ammonia, treated with basic lead acetate, and the filtrate 

 acidified and extracted with ether. The extract was re-treated with 

 basic lead acetate and re-extracted with ether, when crystals of 

 _^-aminobenzoic acid, m.p. 186-4° C., separated from the final extract. 

 On acetylation of the mother-liquors, crystals of ^-acetylaminobenzoic 

 acid (m.p. 259-5° C.) were obtained. Altogether the equivalent of i-6 

 mg. of _/)-aminobenzoic acid was isolated from i kg. of yeast, about 

 one-half that estimated to be present. The extraction was repeated 

 on a sample of plasmolysed yeast heated at 80° C. to destroy enzymes 

 and on another sample of the same yeast which had been allowed to 

 autolyse. From these two samples, ^-acetylaminobenzoic acid equi- 

 valent to 2-7 and 4-7 mg. of ^-aminobenzoic acid per kg. was isolated, 

 corresponding to 57 and 60 % of the amounts estimated to be present 

 in these two preparations. Thus _^-aminobenzoic acid appeared to 

 exist in yeast in combined form, from which it was released on 

 autolysis. 



This was confirmed by Ratner et al.^ who, by a process involving 

 precipitation with a silver salt, fractionation of the lead and bariiun 

 salts and precipitation from alcohol-ether or alcohol-acetone solutions, 

 isolated from 50 kg. of dried yeast, 400 mg. of a polypeptide containing 

 8 % of ^^-aminobenzoic acid. This had no anti-sulphonamide activity, 

 but free ^-aminobenzoic acid was liberated on hydrolysis with acid or 

 alkali. The peptide contained a chain of ten or eleven glutamic acid 

 residues, to which the ^-aminobenzoic acid was attached through its 

 carboxyl group.* Thus the conjugate was analogous in structure to 

 vitamin Be conjugate (page 464). It accounted for 20 to 30 % of the 

 total j!)-aminobenzoic acid content of yeast. 



References to Section 2 



1. S. D. Rubbo and J. M. Gillespie, Nature, 1940, 146, 838. 



2. K. C. Blanchard, /. Biol. Chem., I94i» 140, QiQ- 



3. S. Ratner, M. Blanchard, A. F. Coburn and D. E. Green, ibid., 



1944, 155, 689. 



4. S. Ratner, M. Blanchard and D. E. Green, ibid., 1946, 164, 



691. 



548 



