238 C. B. ANFINSEN [13 



CONCLUSIONS 



It may be stated with reasonable certainty that the polypeptide chain of 

 ribonuclease, free of cross linkages, is not sufficient for catalytic activity 

 since the oxidized and reduced forms of the protein are completely inert. 

 This conclusion is supported by the results of studies which indicate that 

 the methods employed for the cleavage of disulfide bridges do not chemi- 

 cally modify amino acids other than cystine. (One must, however, keep in 

 mind the possibiUty that the open chain does possess intrinsic enzyme acti- 

 vity but that the formation of the proper catalytic configuration is prevented 

 by the presence of sulfonic acid, carboxymethyl, or sulfhydryl groups.) 



The weight of evidence now suggests that there exists in the native mole- 

 cule a constellation of amino acid residues, considerably less in size than 

 the intact protein, which is responsible for catalytic activity. The results 

 of Richards^^ on the limited degradation of the chain with subtilisin, make 

 it likely that the A^-terminal portion of the molecule is not involved. Simi- 

 larly, the last few residues at the C-terminal end of the chain appear to be 

 superfluous. 21* Partial reduction of disulfide bridges^ also seems to be per- 

 missible. 



The physical studies discussed above have indicated a correlation between 

 activity and the presence of a unique type of spectrum, characteristic of 

 tyrosine residues whose hydroxyl groups seem to be linked through hydro- 

 gen bonding to carboxylate groups elsewhere in the molecule. It cannot, 

 of course, be stated that such residues are necessarily implicated in the 

 actual catalytic process. However, it does seem likely that one or more 

 hydrogen-bonded tyrosine residues must be intimately concerned with the 

 'active center' of ribonuclease and that these exist in such a linkage when 

 the geometry of the 'center' is in its proper configuration. 



These chemical and physical observations, when considered together with 

 the fact that limited pepsin digestion causes complete inactivation after the 

 removal of only a small C-terminal fragment,^^ lead us to conclude that 

 at least a part of the catalytically active portion of the ribonuclease chain 

 resides near the C-terminus. The apparent requirement for at least two 

 intact disulfide bridges suggests that such an area of the molecule is joined 

 in close proximity to one or more other essential amino acid sequences. 

 The size and location of these can obviously not be considered until a great 

 deal of further information is available on the relative importance of the 

 individual disulfide bridges and on the extent to which the ribonuclease 

 molecule can be further degraded without complete loss of function. 



* The word 'superfluous' must be used in a very qualified sense, since it appears rather 

 unlikely that 'superfluous' structures would have survived the attrition of mutation and 

 selection that has been at work since Nature first devised ribonuclease molecules if some 

 selective advantage were not involved. 



