STRUCTURAL BASIS OF RIBONUCLEASE ACTIVITY 



137 



Sedimentation constants 



Fig. 8. Sedimentation studies on native ribonuclease and on the enzymatically 

 active derivative obtained by subtilisin digestion (after Richards, 1955). 



that of native ribonuclease although after oxidation one observes somewhat less 

 concentration dependence, though the value at infinite dilution appears to be 

 the same as that found for oxidized native ribonuclease. In further attempts to 

 account for the inactivation of ribonuclease by pepsin in the absence of serious 

 structural change, we have studied the production of small non-protein nitro- 

 gen fragments during this rapid process. By such techniques it has been found 

 that the tetrapeptide, -asp.ala,ser.val, is produced at a rate quantitatively 

 correlated with the rate of inactivation of the enzyme (Anfinsen, 1956). The 

 possibility exists that peptide fragments other than this tetrapeptide (Fig. 12) 

 also appear during the rapid inactivation, although the detection of such fur- 

 ther materials has not as yet been possible. It seems likely, however, that pep- 

 sin has not attacked the protein at some internal point since the physical prop- 

 erties of the inactive derivative, following oxidation of S — S bridges, are 

 extremely similar to those of the oxidized native molecule. 



In summary, we must conclude that pepsin has inactivated ribonuclease by 

 an extremely restricted modifying action which does not at first sight appear to 

 involve internal cleavage of the protein chain. 



It was shown earlier by Shugar (1952) that pepsin digestion of ribonuclease 

 led to changes in the ultraviolet absorption spectrum of the molecule which are 

 characteristic of the rupturing of hydrogen bonds of the sort which appear to 

 exist between tyrosine hydroxyl groups and aspartyl or glutamyl carboxyl 

 groups. These observations prompted an examination of the UV spectrum dur- 

 ing the extremely restricted pepsin treatment described above. It has been found 

 that a shift does indeed occur which is of the order to be expected for the rupture 

 of one such bond (Anfinsen and Tritch, unpublished experiment). This spectral 

 change occurs at a rate which parallels both the rate of appearance of the tetra- 

 peptide fragment and the rate of ribonuclease inactivation. Whether or not the 

 indicated hydrogen bond plays a role in the workings of the active center is, of 



