54 C. H. W. HIRS, M. HALMANN, J. H. KYCIA 



repeating an experiment of the kind illustrated in Fig. 3 a sufficient number 

 of times. The protein was freed from contaminating chloride ion and 

 subsequently oxidized with performic acid under conditions identical to 

 those used in structural work with ribonuclease A [15]. The performic 

 acid-oxidized ribonuclease A derivative was subjected to quantitative 

 amino acid analysis, the results of which demonstrated that complete 

 oxidation of the disulphide bonds had occurred, that methionine had been 

 quantitatively converted to the sulphone, and that there had been no 

 destruction or alteration [15] of tyrosine during the oxidation. The oxidized 

 protein derivative was submitted to tryptic hydrolysis at pH 7 in the 

 presence of catalytic quantities of trypsin prepared by the activation of 

 chromatographically purified [16] chymotrypsinogen-free trypsinogen. 

 The conditions were similar to those already described for the tryptic 

 hydrolysis of performic acid-oxidized ribonuclease A [17]. The mixture 

 of peptides formed after 24 hours of hydrolysis was fractionated on Dowex 

 50-X2 columns in the sodium form by procedures very similar to those 

 described for the tryptic peptides from oxidized ribonuclease A [17]. 

 Formate and acetate buffers were used to facilitate quantitative amino 

 acid analysis of the peptide fractions (cf. [18]). 



All the peptides found in a tryptic hydrolysate of oxidized ribonuclease 

 A [17] were present in the hydrolysate with the exception of peptides O- 

 Tryp 9 and 0-Tryp 14. Identification of the peptide fractions was in each 

 instance confirmed by quantitative amino acid analysis. In order to make 

 certain that ninhydrin-negative peptides were not being overlooked, the 

 aliquots removed from the efiluent fractions were subjected to alkaline 

 hydrolysis [17] prior to ninhydrin analysis. Within the precision attain- 

 able by quantitative analysis of appropriate cuts from the peptide zones 

 on the chromatogram it was clear that the peptides in the hydrolysate 

 of the oxidized, inactive derivative were formed in the same yields as 

 they are from oxidized ribonuclease A. It was not possible to detect 

 any dinitrophenyl peptides present in the tryptic hydrolysate in the 

 effluent from the Dowex 50-X2 column. Moreover, since subsequent 

 extraction with o • i n NaOH failed to remove any significant quantities 

 of material absorbing at 360 m/x from the resin, it is possible that severe 

 "tailing" was responsible for the loss of the dinitrophenyl peptides. The 

 high affinity of dinitrophenyl derivatives of amino acids for the poly- 

 styrene matrix of the resin has already been described. 



Peptides 0-Tryp 9 and 0-Tryp 14 are related [19] in the amino acid 

 sequence of ribonuclease. Peptide O-Tryp 14 is an intermediate in the 

 tryptic hydrolysis of oxidized ribonuclease A. On further hydrolysis it 

 breaks down into peptides 0-Tryp 9 and 0-Tryp 7 (Asp . Arg) by cleavage 

 at the carbonyl bond of the arginine residue of peptide 0-Tryp 7. The 

 absence of peptides 0-Tryp 9 and 0-Tryp 14 in the Dowex 50-X2 eluate 



