236 C. B. ANFINSEN [13 



(c) Heat, alkali, and methylation. Ribonuclease may be progressively inacti- 

 vated, in an irreversible way, by exposure to heat, to alkalinity above pH 

 12'3-12*7,^* and by esterification of free carboxyl groups. These treatments 

 all lead to a shift in spectrum, the extent of the shift being proportional to 

 the extent of inactivation. The titration studies of Tanford and Hauenstein^' 

 indicate that the irreversible change in spectrum occurring at high pH in- 

 volves the modification of approximately 3 hydrogen-bonded tyrosine resi- 

 dues, as suggested also by the studies of Harrington and Schellman^' on 

 the urea denaturation of ribonuclease. In the latter case, however, the shift 

 in spectrum is reversible and is prevented by anions.^* 



{d) Acid, subtilisin digestion, oxidation, and reduction. Scheraga^^ has shown 

 that a spectral shift takes place when ribonuclease is dissolved in acid solu- 

 tions and that the extent of the shift is pH dependent in a manner consistent 

 with the titration of acid groups having pK values in the neighborhood of 

 pH 2. These unusually acid groups may represent the electronegative groups 

 involved in hydrogen bond formation with the tyrosine hydroxyl groups 

 mentioned above, 



Performic acid oxidation, which causes complete inactivation, ^^ produces 

 an irreversible shift in spectrum. ^^ Partial reduction, however, when carried 

 to a point where approximately one disulfide bridge per molecule has been 

 cleaved, and where no trace of remaining native enzyme can be detected 

 electrophoretically, yields a product with essentially full activity and which 

 shows a 'native' spectrum. ^ 



Subtilisin digestion causes extensive and random digestion of the native 

 enzyme and during this digestion there occur parallel changes in enzyme 

 activity and in the degree to which the spectrum has been shifted to lower 

 wavelengths. 



(e) Guanidine hydrochloride ^^ Guanidine hydrochloride is known to be a 

 much stronger denaturing agent than urea (see, for example,^"). It was 

 found that 6m solutions of this salt completely inactivated ribonuclease in 

 contradistinction to the results with urea. The studies with guanidine indi- 

 cated that the substrate molecule was unable to bring about refolding of 

 the polypeptide chain. Spectrophotometric, optical rotatory, and viscometric 

 measurements bear out this conclusion (see Table 6). Optical dispersion 

 measurements (Table 6 and Fig. 9) also indicate that guanidinium ions cause 

 an even greater disorientation of the secondary structure of ribonuclease 

 than does urea, as shown by a comparison of the rotatory dispersion con- 

 stants for the protein in these two systems. In view of these results it was 

 of interest to determine the level of guanidine hydrochloride at which ribo- 

 nuclease is active. Since the activity of ribonuclease is strongly affected by 

 the ionic strength of the assay solution, ^^ and since guanidine hydrochloride 

 is a strong salt, it was first necessary to determine whether or not this agent 



