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. Fischei , ^ 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 

 well as Van Slyke's methods.* Sorensen's method has decided advan- 

 tages in ease of manipulation and accuracy when properly employed. 



Theoretical Discussion oj 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 sho'wii bj' Hugo 

 Schiff. * The formaldehyde converts the radical 



COOH COOH 



= C< into =C< 



NHo N . CH2 



Neutral in Acid in 



reaction reaction 



For example : — 



NH2 N . CH2 



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



COOH COOH 



alanine neutral methylene- water 



formaldehyde alanine . 



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

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

 solution in accordance with the following equation : — 



N . CHo N . CH2 



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



COOH COOK 



That is, a simple neutralizing reaction. 



' Ann. (Leipzig), 169, 150, 1873. 



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



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



« Abderhalden, E. Lehrbuch d. Physiologischen Chemie, Tail 1, 307-652, 1914. 



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



^ Sorensen, S. P. L. Comptos Rendus 7me. Ire Livraison, 1907. 



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



8 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. 



