166 PTEROYLGLUTAMIC ACID 



creas digestion apparently possesses the same activity for the assay orga- 

 nism as PGA. Pteroylheptaglutamate is only 30% hydrolyzed by chicken 

 pancreas and 75 % by hog kidney conjugase. A stepwise combination of the 

 two enzymes accomplishes complete hydrolysis of heptaglutamate com- 

 pound. 



Hog kidney conjugase is readily inhibited by thymus nucleic, yeast nu- 

 cleic acid, and extracts of yeast and molasses (Mims et al}^). A number of 

 proteins such as casein, gelatin, egg albumin, and crystalline bovine plasma 

 albumin also inhibit hog kidney conjugase.^^ 



Thymus and yeast nucleic acids which inhibit hog kidney conjugase have 

 no effect on chicken pancreas conjugase.^^ This enzyme is, however, in- 

 hibited by a glutamic acid polypeptide of p-aminobenzoic acid derived from 

 yeast .2^ p-Aminobenzoyl-7-glutamyl-7-glutamylglutamic acid also inhibits 

 chicken pancreas conjugase. ^^ The tetraethyl ester of this peptide gives no 

 inhibition, and the p-nitro derivative of the acid peptide is half as active 

 as the peptide with an amino group. 



h. Digestion Procedures for Assay Purposes 



Bird et al}^ described four methods for the enzymatic hydrolysis of 

 pteroylglutamic acid conjugates and compared the results with those ob- 

 tained by assay with chicks. These results, presented in Table VIII show 

 that enzymic hydrolysis followed by microbiological assay gave essentially 

 the same results as assay with chicks except in two samples. One sample of 

 liver extract, which gave only half as much pteroylglutamic acid by diges- 

 tion with acetone-desiccated hog kidney and microbiological assay as it 

 did with chick assay, was found to give comparable results when digested 

 with an extract of unheated almond. Certain natural materials were found 

 to contain enzyme inhibitors, and in these cases very large amounts of 

 enzyme were necessary to secure maximum hydrolysis of the conjugates 

 present. 



Digestion with acetone-dried or fresh chicken pancreas at pH 7.2 is the 

 recommended procedure of the Association of Official Agricultural Chem- 

 ists.^® Enzyme digestion is preceded by autoclaving the finely ground sample 

 at the same pH to extract the factor from the tissue. 



21 V. Mims, M. E. Swendseid, and O. D. Bird, /. Biol. Chein. 170, 367 (1947). 



22 A. Z. Hodson, Arch. Biochem. 16, 309 (1948). 



23 E. S. Sims and J. R. Totter, Federation Proc. 6, 291 (1947). 



2" E. L. R. Stokstad, J. Pierce, T. H. Jukes, and A. L. Franklin, Federation Proc. 7, 



193 (1948). 

 26 0. D. Bird, B. lirosslor, R. A. lirovvn, C. J. Canii)l)ell, and A. I). ICnimett, J. Biol. 



Chem.159, 631 (1945). 

 26 Methods of Analysis, .Association of Offici.'il Agricultural Chemists, 7th ed., p. 



784, 1950. 



