94 PTEROYLGLUTAMIC ACID 



later shown to be pteroyltriglutamic acid, was isolated by Hutchings et al}^ 

 from a cell-free filtrate obtained from the aerobic fermentation of a Coryne- 

 haderium. The method involved adsorption and elution, esterification of 

 the barium salt to give the methyl ester, and extraction of the ester from an 

 aqueous solution with butanol. The methyl ester at this stage could be 

 dissolved in hot methanol and reprecipitated on cooling in the presence of 

 0.05 N sodium chloride. In the absence of an electrolyte a gel was formed 

 instead. By repeated reprecipitation of this ester from methanol in the 

 presence of an electrolyte, it was obtained in a microcrystalline form. The 

 free acid was obtained by hydrolysis of the ester and crystallization at pH 

 2.8 in the presence of electrolytes such as sodium or calcium chloride. 



Although pteroylglutamic acid can be precipitated from water and 

 methanol in the absence of electrolytes, pteroyltriglutamic acid and its 

 ester require the presence of electrolytes for their precipitation. The solu- 

 bility of pteroyltriglutamic acid in the presence of calcium chloride at pH 

 2.8 is 3.0 mg. per millihter at 80° and 0.10 mg. per milliliter at 5°. 



Binkley et al}"^ • ^^ isolated in crystalline form a microbiologically inactive 

 conjugate of PGA (vitamin Be conjugate) from yeast. These workers found 

 that a yeast extract rich in anti-anemic activity for the chick was relatively 

 inactive for L. casei or S. faecalis. Digestion of this yeast concentrate with 

 a suitable enzyme from hog kidney increased the microbiological activity. 



This conjugate which crystallized from 5 % sodium chloride solution was 

 0.4 % as active as PGA for L. casei and 0.2 % as active for S. faecalis assay. 

 This conjugate was later found by Pfiffner et ai.'^ to contain seven glutamic 

 acid residues. Enzymes which split this conjugate have no effect on its 

 methyl ester, showing that the conjugases may be classified as carboxypep- 

 tidases. Although PGA exists in a microbiologically inactive form in liver, 

 no conjugates have yet been isolated from this source. A more detailed dis- 

 cussion of the various types of PGA precursors occurring in liver will be 

 given in a subseciuent portion of this review. 



a. Pteroylglutamic Acid-Protein Complex in Yeast 



Specific and reversible combinations of proteins and vitamins have l)een 

 noted in many instances in which the vitamin forms the basic part of the 

 coenzyme. This has been demonstrated for thiamine, pyridoxal, riboflavin, 

 and nicotinamide. Attempts have been made to find similar combinations 



16 B. L. Hutchings, E. L. R. Stokstad, N. Bohonos, N. H. Slojuie, and Y. SubbaRow, 

 J. Am. Chem. Soc. 70, 1 (1948). 



1' S. B. Binkley, O. D. Bird, E. S. Bloom, R. A. Brown, D. G. Calkins, C. J. Camp- 

 bell, A, D. Emmett, and J. J. Pfiffner, Science 100, 36 (19-44). 



18 J. J. Pfiffner, D. G. Calkins, B. L. O'Dell, E. S. Bloom, R. A. Brown, C. J. Camp- 

 bell, and O. D. Bird, Science 102, 228 (1945). 



13 J. J. Pfiffner, D. G. Calkins, E. S. Bloom, and B. L. O'Dell, /. Am. Chem. Soc. 68, 

 1392 (1946). 



