162 PTEROYLGLUTAMIC ACID 



with zinc in 0.5 A^ hydrochloric acid and the aromatic amine estimated by 

 the method of Bratton and Marshall.^ In crude reaction products contain- 

 ing free p-aminobenzoic acid or its derivatives, the aromatic amine is de- 

 termined before and after reduction with zinc. The increase resulting from 

 reduction is a measure of the amount of PGA. p-Aminobenzoic acid and 

 its peptides with other amino acids give the same amount of color on a 

 molar basis when diazotized and coupled with A^-(l-naphthyl)ethylene- 

 diamine dihydrochloride by the method of Bratton and Marshall. Thus the 

 method can be used for pteroic acid, its amide, ester, or peptide with any 

 amino acid. Any derivative which contain? a substitution on the nitrogen 

 of the p-aminobenzoic acid cannot be estimated by this method because 

 the presence of a primary amino group is necessary for the diazotization 

 and coupling reaction. 



Titanous chloride has been used by Glazko and Wolf^ to reduce pteroyl- 

 glutamic acid. They found that, in the assay of tissue homogenates by the 

 zinc reduction methods, adenine and nucleic acid products interfered by 

 producing diazotizable substances on reduction with zinc. The use of 

 titanous chloride eliminates this difficulty and permits the analysis of 

 PGA in the presence of adenine. 



A micromethod has been proposed by Allfrey et al} which is based on 

 the oxidation of pteroylglutamic acid by alkaline permanganate solution 

 to give 2-amino-4-hydroxypteridine-G-carboxylic acid. This pterin is then 

 estimated by fluorometric methods. In the presence of extraneous fluores- 

 cing pigments, such as xanthopterin, which are affected by permanganate, 

 the 2-amino-4-hydroxypteridine-6-carboxylic acid is adsorbed on Florisil, 

 eluted, and then measured fluorometrically. This procedure has been 

 adopted to use with natural products and gives results which are com- 

 parable to, although slightly higher than, those obtained by microbiological 

 methods. Such a chemical procedure has the advantage that it measures 

 total soluble pteroylglutamic acid in free and in conjugated form. 



B. MICROBIOLOGICAL METHODS 



Two main problems are involved in the microbiological estimation of 

 ptei'oylglutamic acid in natural products. The first invoh-es extraction and 

 hydrolysis of pteroylglutamic acid conjvigatcs which may be present; the 

 other, assay of the microbiologically active compounds. Detailed descrip- 

 tions of these methods in review form have been published by Stokstad 

 and llulchings*' and Snell.'^ 



3 A. C. Bratton and E. K. Marshall, Jr., /. Biol. Chem. 128, 537 (1939). 



•' A. J. Glazko and L. M. Wolf, Arch. Biochem. 21, 241 (1949). 



5 V. Allfrey, L. J. Teply, C. Geffen, and C. G. King, J. Biol. Chem. 178, 465 (1949). 



^ E. L. R. Stokstad and B. L. Hutchings in Biological Symposia, The Microbiolog- 



