13] STRUCTURE & ENZYMATIC ACTIVITY OF RIBONUCLEASE 235 

 Other data in Table 6 indicate, however, that refolding by anions is not 

 quantitatively complete. Thus measurements of the optical rotatory properties 

 and the intrinsic viscosity of ribonuclease in 8m urea solutions contain- 

 ing orthophosphate or polymetaphosphate ions give values that are com- 

 patible with a still partially unfolded form of the protein. The combined 



X 



2800 2900 



WAVE LENGTH. A 



Fig. 8. Decreases in extinction, compared with native ribonuclease in water, of (A), 

 inactive derivative produced by limited pepsin digestion, (B), ribonuclease in 8m urea, 

 and (C), ribonuclease in 8m urea containing 003m orthophosphate ions. Curves calcu- 

 lated from individual curves obtained on Cary ultraviolet recording spectrophotometer. 2» 



data suggest that that portion of the molecule which is responsible for the 

 modified light absorption of native ribonuclease is also concerned with 

 catalytic activity and that catalysis is unimpaired in spite of minor structural 

 alterations elsewhere in the fabric of the protein. 



(b) Pepsin digestion. This conclusion is further supported, in a converse sort 

 of way, by the spectrophotometric and optical rotatory results obtained on 

 the derivative produced by limited pepsin digestion. ^^-^^ This derivative, 

 which, as was discussed above, differs from the native enzyme only by 

 lacking the four C-terminal residues, shows a shifted spectrum (curve A, 

 Fig. 8) similar to that seen in 8m urea. Orthophosphate is without effect on 

 this pepsin-induced shift. The specific optical rotation of the derivative is 

 similar to that obtained with native ribonuclease or with ribonuclease in 

 8m urea solutions containing polyvalent anions. It may be tentatively con- 

 cluded, therefore, that the secondary structure of the derivative is reasonably 

 'native', a conclusion also consistent with earlier estimates of the intrinsic 

 viscosity and sedimentation behavior of this material. ^^ 



