VI. ESTIMATION 165 



heptaglutamatc derived from yeast (Pfiffner cl al."). Evidence that hog 

 kidney conjugasc consists of two enzymes has been ol^tained by Mims and 

 Bird."* The liydrolysis of pteroylheptagkitamic acid was found to reciuire 

 botli components, whereas the hydrolysis of pteroyltriglutamic acid could 

 be effected by only one. Calcium ion has not been found to be a part of the 

 hog kidney conjugase system. 



Conjugases have been observed by Simpson and Schweigert^^ in the blood 

 of several animal species, including man. AVhen the conjugase in blood is 

 allowed to act on the conjugates in blood, the pH optimum is observed to be 

 7. HoweA'er, when the substrate is either pteroylheptaglutamic acid or the 

 conjugates present in taka-diastase, equal activity is obtained with blood 

 conjugase at pH 4.5 and at pH 7. This suggests that the conjugases in blood 

 have different pH optima, depending on the substrate. These observations 

 point out the hazards associated with the use of crude enzyme preparations 

 in the hydrolysis of conjugates prior to microbiological assay. Incubation 

 of taka-diastase with blood produces an increase in pteroylglutamic acid 

 acti\-ity which has been interpreted as indicating the presence of conjugates 

 in the blood which are hydrolyzed by conjugases in the taka-diastase. Ac- 

 tually, the converse has proved to be true, and the conjugases in the blood 

 are really hydrolyzing the conjugates present in the blood and in the taka- 

 diastase. 



Complete hydrolysis of the pteroylheptaglutamate cannot be accom- 

 plished by either chicken pancreas or hog kidney conjugase alone. Thus 

 Sreeni\'asan et a/.-"^ found that complete hydroh'sis of conjugates for assay 

 purposes could be attained only by the stepwise use of chicken pancreas 

 and hog kidney conjugase. Rat liver contains two enzymes with pH optima 

 at 4.5 and 7. Autolysis of liver or digestion of hver by hog kidney conjugase 

 at pH 4.5 hberates very httle PGA. Since hog kidney conjugase produces 

 approximately 75 % hydrolysis of the pteroylheptaglutamate, it is apparent 

 that this conjugate is not present in liver. Autolysis or chicken pancreas 

 digestion of liver at pH 7.0 produces an increase in activity. Further evi- 

 dence for the differences in the behavior of these two conjugases lies in the 

 fact that pteroyltriglutamic acid is completely split by both chicken pan- 

 creas and hog kidney conjugases where microbiological activity is used as a 

 criterion of hydrolysis. The pteroyldiglutamic acid formed by chicken pan- 



'^ J. J. I'fiffncr, S. B. Binkley, E. S. Bloom, and B. L. O'Doll, ./. Am. Chem. Soc. 69, 

 1476 (1947). 



1* V. Mims and O. D. Bird in Vitamin Sj-mposium, Conference of American Associa- 

 tion for the Advancement of Science, Colli}' College, 1948; cited by M. Laskowski, 

 Ann. Rev. Biochem. XIX, 21 (19.50). 



" R. E. Simpson and B. S. Schwoigort, Arch. Biochem. 20, 32 (1949). 



20 A. Sreenivasan, A. 10. Harper, and C. A. Eivehjcm, J. Biol. Chcm. 177, 117 (1949). 



