152 MASS. EXPERIMENT STATION BULLETIN 167. 



Methods for Quantitative Determination of Proteolysis. 



Previous to the recent development of protein chemistry and methods 

 for the determination of cleavage products of proteins, the quantitative 

 determination of proteolysis was not successful. With Hlaswetz and 

 Habermann's ^ successful researches, and the investigations of Kossel and 

 Kutscher, ^ together with the remarkable findings of E. Fischer, ^ Abder- 

 halden, ^ Plimmer^ and other recent workers, the chemical nature of pro- 

 teins began to assume more definite form. Then several methods for 

 determining cleavage products were advanced. Since the proteolysis is 

 mainly the hydrolytic splitting of proteins, the degree of proteolysis can 

 be followed quantitatively by determining the amount of the cleavage 

 products from time to time. It is most important for our purpose to find 

 a method which enables us to determine the amino acids as a group, 

 because we naturally expect the amino acids as extended cleavage products. 

 Sorensen's formol titration method* has been selected for this purpose. 

 This has been done only after reviewing Hausmann and Osborn's,^ as 

 weU as Van Slyke's methods. ^ Sorensen's method has decided advan- 

 tages in ease of manipulation and accuracy when properly employed. 



Theoretical Discussio7i of Formol Titration. — The reaction taking place 

 between formaldehyde and amino acids, as well as its application to the 

 quantitative determination of amino acids, was first shown by Hugo 

 Schiff. ' The formaldehyde converts the radical 



COOH COOH 



= C< into =C< 



NH2 N.CH2 



Neutral in Acid in 



reaction reaction 



For example : 



COOH COOH 



NH. N . CHo 



CH3 . CH< +HCOH = CH3 . CH< +H2O 



alanine neutral methylene- water 



formaldehyde alanine 



That is, the addition of neutral formaldehyde to the practicallv neutral 

 solution of alanine, which can be titrated with the standardized alkaline 

 solution in accordance with the following equation ; — 



N . CH2 N . CH2 



CH3 . CH< +KOH =CH3 . CH< +H2O 



COOH COOK 



That is, a simple neutralizing reaction. 



» Ann. (Leipzig), 169, 150, 1873. 



« Zeit. Physiol. Chem., 22, 176, 1896-97; 25, 165, 1895. 



* Untersuchungen iiber Aminosauren, Polypeptide und Proteins, Berlin, 1906. 



* Abderhalden, E. Lehrbuch d. Physiologisohen Chemie, Teil 1, 307-652, 1914. 



' Plimmer, R. H. The Chemical Constituent of the Proteins, Monograph on Biochemistry. 



* Sorensen, S. P. L. Comptes Rendus 7me. Ire Livraison, 1907. 

 ' Zeit. Physiol. Chem., 27, 95, 1899; 29, 136, 1900. 



« Proc. Soc. Exp. Biol, and Med., 7, 46, 1910; Berichte d. D. Chem. Ges., 43, 3170, 1910. 

 » Schiff, H. Ann. der Chemie, 310, 25, 1899; 319, 59 et 289, 1901; 325, 348, 1902. 



