12] STRUCTURE OF RIBONUCLEASE 219 



of aspartic and glutamic acid, the experiments with these enzymes have in 

 certain instances also furnished information about the sequences of some 

 of the residues in the peptides. The interpretation of the results, however, 

 is not always clear-cut, since, as was noted above, partially degraded pep- 

 tides may move on the Amberlite IR-120 columns at rates identical with 

 or close to those for free amino acids. 



The application of these procedures to 17 of the 23 peptides isolated for 

 sequence studies has given the detailed information shown in Fig, 2, which 

 may now be discussed in more detail. The sequence o'f the first ten residues 

 in the peptide chain was established earlier; the first four as a result of Dr 

 Anfinsen's end-group analyses on the intact protein* and the rest as a con- 

 sequence of our analyses of the peptides formed by the action of trypsin 

 and chymotrypsin.^-'' The sequence has now been independently confirmed 

 by the use of carboxypeptidase, and by step-wise degradation. More recent 

 work has revealed the sequence from the 11th to the 31st residues shown 

 in Fig. 2. The sequence Ser.Arg.Asp(NH2).Leu.Thr.Lys.Asp.Arg starting 

 at the 32nd residue was previously worked out by Redfield and Anfinsen,^^ 

 and has been confirmed in the present work. Thus the arrangement of the 

 first 39 residues in the chain is now known. The portion of the chain from 

 the 40th to the 61st residue is accounted for by the tryptic peptide 0-Tryp 9, 

 containing 22 residues, on the sequence of which work is in progress. From 

 the sixth lysine residue in the chain (residue 61) the sequence shown in 

 Fig. 2 passes through the pentapeptide 0-Tryp 5, which was considered in 

 some detail before, and continues through asparagine and glycine for a 

 total of 44 residues. The latter is the longest sequence of residues worked 

 out to date along the peptide chain of ribonuclease. Finally, the work of 

 Dr Anfinsen and his colleagues ^^ has estabhshed the sequence of the four 

 last residues in the chain. 



Concurrently with these studies, Dr Spackman in our laboratory, ^^ and 

 Ryle and Anfinsen at Bethesda,^' have been working on the positions of 

 the disulfide bonds in ribonuclease. The approach in both laboratories is 

 similar in several respects to the procedures used so successfully by Ryle, 

 Sanger, Smith, and Kitai,^^ in the elucidation of the disulfide structure 

 present in insulin. 



Ribonuclease in which the disulfide bonds are intact is not hydrolysed 

 in aqueous solution by either trypsin or chymotrypsin. In the presence of 

 2 M guanidinium chloride, however, successive hydrolysis at pH 7-5 by 

 trypsin and chymotrypsin, followed by desalting and chromatography on 

 Dowex 50-X2, gave the result shown on the top part of Fig. 5. Enzymatic 

 action was carried out in the presence of A^-ethylmaleimide to abolish or 

 at least minimize the disulfide interchange reaction. The cystine containing 

 peptides, labeled A to F in the curve, were estimated by the phosphotung- 

 state procedure of Kassell and Brand, ^^ the results of which are shown by 

 the open circles. The fractions in the major peaks were pooled, desalted, 



