130 C. B. ANFINSEN AND R. R. REDFIELD 



The studies I wish to discuss this morning are concerned with the enzyme, 

 ribonuclease, an enzyme which possesses very favorable structural and enzy- 

 matic properties for investigations of this sort. The detailed sequence of ribo- 

 nuclease has been under investigation both in our own laboratory and by Drs. 

 Hirs, Moore and Stein at the Rockefeller Institute in New York. The ap- 

 proaches to the structure of this protein used in the two laboratories have been 

 quite dissimilar and it is very gratifying to all of us concerned that the present 

 picture of the ribonuclease chain is compatible with both sets of results (Anfin- 

 sen, Redfield, Choate, Page and Carroll, 1954; Hirs, JNIoore and Stein, 1956; 

 Redfield and Anfinsen, 1956; Hirs, INIoore and Stein, 1956). A discussion of the 

 research involved in establishing this structural picture is beyond the scope of 

 the present discussion. Therefore, I can do no more at this time than to show 

 Fig. 1, which gives the alignment of amino acids in the ribonuclease molecule 

 according to our own data at the present time (revised as of May 1, 1956). I 

 could equally well have shown an illustration of the Rockefeller data which 

 shows considerably more detail in some areas and less in others. While your at- 

 tention is on this figure, I would like to indicate to you the following points 

 which will be of interest in the subsequent discussion. Ribonuclease consists of 

 a single peptide chain containing 124 amino acid residues. It contains eight half- 

 cystine residues, all of which are joined in disulphide bonding (Anfinsen et al, 

 1954; Rabinovitch and Barron, 1955). Inspection of the formula indicates that 

 the N-terminal end of the molecule contains a long non-disulphide linked "tail", 

 and as is more apparent from the structure as drawn from the data of Hirs, 

 INIoore, and Stein, a fairly long C-terminal "tail" as well. 



The physical properties of ribonuclease are of particular interest in our pres- 

 ent discussion. Ribonuclease has a molecular weight of 14,000 and from meas- 

 urements of its diffusion constant and sedimentation constant, as well as its 

 intrinsic viscosity, one can state that the molecule exists in solution as a rather 

 compact, globular, protein (Anfinsen et al, 1954). Osmotic pressure studies 

 carried out by Dr. Donald Kupke (personal communication) at the Carlsberg 

 Laboratories have established that the protein is monodispersed under a variety 

 of conditions, giving it a distinct advantage over insulin in studies relating struc- 

 tural and physical properties since the polymerization of insulin is strongly 

 dependent both on pH and concentration. Oxidation of ribonuclease with per- 

 formic acid, which leads to the rupture of the S — S bridges, results in the pro- 

 duction of an inactive, oxidized derivative (Anfinsen et al, 1954) having the 

 predicted molecular weight (i.e., slightly larger than the native enzyme), thus 

 indicating the presence of only a single chain. The amino acid analysis of the 

 oxidized derivative agrees with that of the native molecule according to both 

 the Rockefeller Institute studies and to our own. 



Fig. 2 shows data obtained by Harrington and Schellman (1956) on the con- 

 centration dependence of sedimentation constants for both native and oxidized 



