On Determining the Chemical Structure of Proteins 281 



N-terminal position, might or might not be present, because little is 

 known as to whether trypsin can hydrolyze bonds of this type. 



The rate at which oxidized ribonuclease is split by trypsin is shown 

 in Fig. 2. Amino acid analyses of the oxidized preparation employed 

 showed that cystine had been converted quantitatively to cysteic acid 

 and methionine to the sulfone, but that all the other amino acids were 

 untouched. Moreover, end-group analysis by the DNP technique 



Time, hours 



Fig. 2. Hydrolysis of oxidized ribonuclease by trypsin at 7)H 7.0 and 25° ; ribo- 

 nuclease concentration, 0.5%; trypsin concentration, 0.0025%. 



revealed that the peptide chain had not been cleaved to any significant 

 degree during the oxidation. On the basis of the curve shown in 

 Fig. 2, it was decided to investigate the peptides present after both 

 3 and 20 hours of tiyptic action. For this purpose, the hydrolyzate 

 was fractionated on a column (150 X 2 cm.) of Dowex 50-X2 with 

 the results shown in Fig. 3. The experiment was performed with about 

 200 mg. of ribonuclease so as to yield a sufficient quantity of each 

 peptide for further structural investigation. As a first step, the quan- 

 titative amino acid composition of each peptide was determined after 

 acid hydrolysis by chromatography on columns of Dowex 50-X4. 40 

 The buffer salts from the eluent used in the Dowex 50-X2 column 

 usually did not interfere in this process and were not removed. The 

 amino acid composition of each peptide obtained in pure form in the 

 initial fractionation is shown on the curve in Fig. 3. The analyses 



