12] STRUCTURE OF RIBONUCLEASE 215 



acid residues, should result in the cleavage of the bonds indicated by the 

 dashed lines with the formation of three peptides. Of these, one should be 

 a peptide already characterized (O-Chy 11, Fig. 1) in chymotryptic hydro- 

 lysates of oxidized ribonuclease, while the other two should be new pep- 

 tides with amino acid compositions in agreement with those predicted by 

 the formula. The top curve in Fig. 3 shows the separation on a 150 cm. 

 column of Dowex 50-X2 of the three peptides formed upon chymotryptic 

 hydrolysis of peptide 0-Tryp 4. A comparison of the amino acid com- 

 position of these peptides (given adjacent to each of the peaks on the curve) 

 with the partial structural formula in Fig. 1 reveals that the expected frag- 

 ments have indeed been isolated. 



The second curve in Fig. 3 illustrates the corresponding result with pep- 

 tide 0-Tryp 9. In this experiment, the yields (shown in parentheses after 

 each of the peaks on the curve) were not so favorable, because additional 

 cleavage occurred at two bonds which were not markedly attacked when 

 lower enzyme concentrations were used in the experiments with the intact 

 oxidized protein. Finally, the bottom two curves in Fig. 3 show the results 

 obtained upon chymotryptic hydrolysis of peptides O-Tryp 2 and O-Tryp 16. 

 Once again the products predicted from the partial structural formula were 

 formed. These experiments, therefore, not only furnished peptides suitable 

 for further sequence work, but the results also provided strong substantia- 

 tion for the deductions that led to the derivation of the partial formula. 



Once 10 to 20 mg. quantities of 23 peptides of suitable size for sequence 

 work had been prepared from the entire ribonuclease molecule, we were 

 confronted with a more familiar type of problem, namely, that of deriving 

 the amino acid sequence in relatively small peptides. We thus were fortunate 

 in being able to profit from the experience of the many people who have 

 been through this territory before us. A variety of chemical and enzymatic 

 methods has been employed for the determination of the sequences of the 

 amino acid residues in these peptides. The chemical methods have included 

 the DNP procedure of Sanger for iV-terminal residues, hydrazinolysis for 

 the detection of carboxyl-terminal amino acids (by the Braunitzer and 

 Schramm^" modification of the Akabori procedure), and, in particular, step- 

 wise degradation from the amino-end by the phenyl-wo-thiocyanate method 

 of Edman.^^ In the Edman procedure, each step of the degradation has 

 been followed by quantitative amino acid analysis of the peptide remain- 

 ing after removal of the phenylthiohydantoin. 



In our hands, the most effective method for carrying out the reaction of 

 phenyl-/5o-thiocyanate with the free amino groups of peptides has been the 

 one described by Ottesen and Wollenberger^^ in their work on the peptides 

 liberated from ovalbumin during the formation of plakalbumin. Routinely, 

 the reaction is carried out at 40° in 30 per cent aqueous dioxane, and the 

 pH is maintained at 8-0 with the aid of a pH-stat. In this way the reaction 

 may be followed quantitatively. The rate of the reaction is a function of the 



