II. niF.MISTKY 95 



lietwepii PCiA and proteins. Allfroy and King-" found Ihal during fractional 

 procipitalion of a yeast autolyzate with ammonium sulfate there was a 

 correlation between the amounts of PGA conjugate and protein that were 

 precipitated. Tlie P(JA conjugate was measured microl)iologically after 

 appropriate enzyme digestion. The linkage between PCJA conjugate and 

 protein is essentially salt-like or highly dissociable. The bond is broken by 

 the addition of organic solvents such as ethanol, acetone, or dioxane to 

 protein fractions prepared by salting-out procedures. Heating for 1 minute 

 at 80° or for 5 minutes at 60° also released the P(1A conjugate from the 

 protein. During dialysis of freshly prepared yeast autolyzates in viscous 

 casing the PGA conjugate was rapidly released from the protein. By the 

 use of fractional precipitation with ammonium sulfate between 2.4 .V and 

 3.1 M at varying pH values, protein preparations were obtained which 

 contained 160 y of PGA per gram in the form of the microbiologically 

 inactive conjugate. Pteroylheptaglutamic acid by itself in low concentra- 

 tions is not precipitated under these conditions. 



Six crystalline proteins were obtained from this ammonium sulfate pre- 

 cipitate. Most contained little or no PGA activity, the highest having a 

 value of 75 y of PGA per gram. 



3. Degradation Reactions 

 a. Degradation of Pteroyltriglutamic Acid 



The relationship between pteroyltriglutamic acid isolated from a fer- 

 mentation product and PGA isolated from liver was established by Stok- 

 stad et aU^ by the use of anaerobic alkaline hydrolysis. The triglutamic 

 acid derivative, which is active for L. casei but only slightly active for 

 S. faecalis R, was rapidly inactivated for both organisms by aerobic alkaline 

 hj^drolysis. Anaerobic hydrolysis, however, produced only a slight decrease 

 in the activity for L. casei and greatly increased the activity for S. faecalis 

 R. On anaerobic alkaline hydrolysis the ratio of the activity for these two 

 organisms approached that of pteroylglutamic acid isolated from liver. 

 Two moles of a-amino acid nitrogen were liberated during anaerobic 

 alkaline hydrolysis, and the active compound which was formed was ap- 

 proximately^ half as active as pteroylglutamic acid by ])oth L. casei and 

 S. faecalis R assay. This compound was later identified as racemic pteroyl- 

 glutamic acid })y comparison with the synthetic material. The pteroyl- 

 glutamic acid had apparently been racemized by the anaerobic alkaline 

 hydrolysis. 



Stokstad et al}^ found that aerobic alkaline hydrolysis of pteroyltriglu- 

 tamic acid or of racemic PGA resulted in the formation of a fluorescent pig- 



20 V. G. Allfrey and C. G. King, J. Biol. Chem. 182, 367 (1950). 



21 E. L. R. Stokstad, B. L. Hutchings, J. H. Mowat, J. H. Boothc, C. W. Waller, 

 R. B. Angier, J. Semb, and Y. SubbaRow, J. Am. Chem. Soc. 70, 5 (1948). 



