12 



Studies on the structure of ribonuclease 



C. H. W. HIRS, WILLIAM H. STEIN AND 

 STANFORD MOORE 



The Rockefeller Institute for Medical Research, New York 21, N.Y. 



Rather than attempt a review of published information that deals with the 

 structure of ribonuclease (cf. 1, for example), we will in the present dis- 

 cussion emphasize the more recent work and the methods that are cur- 

 rently being employed to bring the structural studies to a conclusion. Some 

 recapitulation is desirable, however, and it can be furnished by reference to 

 the partial structural formula for oxidized ribonuclease shown in Fig. 1, 

 which summarizes the status of our knowledge as of early 1956.^ The single 

 chain of 124 residues in the oxidized protein is portrayed in four segments, 

 which should be imagined as joined end to end. The bonds at which the 

 chain is attacked by proteolytic enzymes are indicated by the vertical lines : 

 solid lines for trypsin, dashed lines for chymotrypsin, and dotted lines for 

 pepsin. The residues included within the parentheses are of undetermined 

 sequences. 



The derivation of the formula shown in Fig. 1 owes very much to the 

 stimulating and pioneering investigations of Sanger and his colleagues on 

 the structure of insulin (cf. 3 for references). Several of the steps in the 

 derivation were patterned directly after his work ; others had to be changed 

 to a greater or lesser extent depending upon the nature of the special 

 problems encountered. A sample of ribonuclease containing 93 per cent of 

 the A component was employed for most of the structural study. The fact that 

 ribonuclease contains but a single peptide chain, and that this chain bears the 

 sequence Lys.Glu.Thr.Ala- at the amino-end, was estabhshed by Anfinsen 

 and his colleagues* by the use of the DNP technique. The first step in our 

 own studies was an amino acid analysis that showed that ribonuclease A 

 contained 124 to 126 amino acid residues.^ This was followed by oxidation 

 of the single chain at low temperature with performic acid^ under condi- 

 tions chosen so as to avoid the formation of chlorotyrosine. 



The four disulfide bonds in the molecule were thus oxidized to eight 

 sulfonic acid groups, and the four methionine residues to the sulfone. The 

 other amino acids were not affected. The oxidized protein was subjected to 



