13] STRUCTURE & ENZYMATIC ACTIVITY OF RIBONUCLEASE 233 



2. PROTEOLYTIC DIGESTION 



As shown by Richards^^ limited subtilisin digestion of ribonuclease causes 

 the cleavage of what appears to be a single peptide bond located within 

 twenty or so residues of the A^-terminal end of the polypeptide chain. ^^ The 

 resulting macromolecular derivative has full enzymatic activity. Degradation 

 of the C-terminal end of ribonuclease with carboxypeptidase pre-treated with 

 diisopropyl fluorphosphonate (DIP) removes the C-terminal valine residue 

 (and a variable amount of the two preceding residues, serine and alanine) 

 without loss of activity. 2^ Non-DIP-treated carboxypeptidase effects a con- 

 siderably greater number of cleavages and still leaves most of the activity 

 even after fairly extensive attack. ^^ 



Limited pepsin digestion at pH 1 -8, on the other hand, causes the com- 

 plete inactivation of ribonuclease at a rate parallel to the rate of release 

 of the C-terminal tetrapeptide sequence. ^^ No indications of rupture of 

 other peptide bonds have been found by a variety of chemical and physical 

 methods. 2* The inactive derivative contains a C-terminal phenylalanine resi- 

 due as would be expected from the sequence data shown in Fig. 3 above. 



3. CORRELATIONS BETWEEN SPECIFIC ASPECTS 

 OF TERTIARY STRUCTURE AND ENZYMATIC 



ACTIVITY 



(a) Urea. In an earlier paper it was suggested that enzymatic activity in this 

 protein is not dependent upon an intact system of hydrogen bonds since 

 activity is undiminished in 8m urea solutions. ^^ Our more recent studies 

 indicate that this conclusion is probably not quantitatively valid. This re- 

 appraisal is necessary in view of the results of experiments in which the 

 marked shift in the spectrum of ribonuclease to lower wavelengths in 8m 

 urea solutions has been studied as a function of the concentration of certain 

 polyvalent anions and polyanions.^^-^^ The curves in Fig. 8, for example, 

 indicate the differences in molar extinction, between native ribonuclease in 

 0-1 M-KCl and in 8m urea (curve B), and in 8m urea containing 0-003m 

 orthophosphate ions (curve C). Low levels of orthophosphate, arsenate, 

 polymetaphosphate, and uridylate (but not citrate) ions cause a complete 

 reversion of the shifted spectrum to that exhibited by the native enzyme 

 in the absence of urea. This reversion is quantitative at concentrations of 

 ion, as low as 0'05m (i.e., about 60-70 atoms of phosphorous/mole of ribonu- 

 clease). A summary of these, and related experiments on ultraviolet absorp- 

 tion characteristics, is given in Table ö.^^-^^-^^ The table includes, for 

 comparison, the results of experiments with monovalent anions, which do 

 not show such an effect except at very high ionic strengths. (It should be 

 stated that the most desirable test, namely an estimate of the refolding 

 capacity of ribonucleic acid itself, is not technically feasible because of the 



