On the Structural Basis of Ribonuclease 

 Activity 



C. B. Anfinsen and R. R. Redfield 



National Heart Institute, National Institutes of Health, Bethesda, Md. 



THE PROBLEM OF THE MOLECULAR BASIS of cnzyme action has, until very 

 recently, been contemplated by biochemists with understandable awe 

 because of the well-entrenched assumption that enzyme action may in- 

 volve the organized participation of the entire integrated molecular structure 

 in the transfer and rearrangement of electrons (Szent-Gyorgyi, 1941; Wirtz, 

 1948; Evans and Gergely, 1949; Geissman, 1949). In addition, knowledge of 

 the details of the covalent structure of enzymes has been so fragmentary that 

 unambiguous assignments of amino acid secjuences and areas to the "active 

 centers" of enzymes has been essentially impossible. The elegant investigations 

 of Sanger and his collaborators (Sanger and Tuppy, 1951; Sanger and Thomp- 

 son, 1953; Ryle, Sanger, Smith and Kitai, 1955) furnished biochemists with the 

 confidence and methodological background for the studies on enzyme structure 

 which are now taking place in many laboratories. As a result of such structural 

 studies and of other investigations dealing with the involvement of the second- 

 ary structures of proteins in their catalytic activity, the view has begun to 

 emerge that the activity of many enzymes may, indeed, be associated with only 

 a relatively small part of the protein molecule (Aniinsen, Harrington, Hvidt, 

 Linderstrjzim-Lang, Ottesen and Schellman, 1955; Richards, 1955; Hill and 

 Smith, 1955; Aniinsen and Redfield, 1956). It has been known for some time 

 that minor modifications could be made in enzymes without loss of activity. 

 Thus, for example, modification of side chains by introduction of iodine atoms 

 or acetyl groups has been shown to be without detrimental effects in some cases. 

 More recently a number of enzymes have been shown to be unaffected by more 

 drastic procedures such as the removal of C-terminal residues and sequences 

 by the proteolytic enzyme, carboxypeptidase, or of N-terminal sequences by 

 leucine-aminopeptidase. The biologically active proteins thus studied include 

 insulin (Harris and Li, 1952), tobacco mosaic virus (Harris and Knight, 1955). 

 ACTH (Harris and Li, 1955), ribonuclease (Kalnitsky and Rogers, 1956), chy- 

 motrypsin (Gladner and Neurath, 1954), and papain (Hill and Smith, 1956). 

 The latter three, ribonuclease, chymotrypsin, and papain, are of particular 

 interest since their activity, after carboxypeptidase or amino-peptidase treat- 

 ment, could be tested in vitro, and the possibilities of resynthesis of the original 

 form during in vivo testing could thus be exlcuded. 



128 



