13] STRUCTURE & ENZYMATIC ACTIVITY OF RIBONUCLEASE 231 

 intermediates. Regeneration of enzymatic activity takes place upon reoxida- 

 tion of the sulfhydryl groups with molecular oxygen and 'fingerprints' (filter 

 paper electrophoretic patterns of subtilisin digests) of the reaction mixture 

 after reoxidation (Fig. 6) suggest that there occurs a partial reformation of 

 the same disulfide bonds as those occurring in the native molecule. ^^ Specific 

 identification of the labile, presumably non-essential, disulfide bonds has 



-Urea 



80 160 



MINUTES 



240 



Fig. 4. The kinetics of reduction of the disulfide bridges of ribonuclease in the presence 

 and absence of 8 m urea.^^ 



SH GROUP 



Fig. 5. Activity of ribonuclease at various stages of reduction (expressed as percentage 

 of the specific activity of native ribonuclease) as a function of the number of moles of 

 sulfhydryl per mole of enzyme. A, Reduction in absence of urea; ^, reduction in 8m urea; 

 □ , reoxidation of fully reduced, inactive ribonuclease; 0> reoxidation of samples con- 

 taining more than six sulfhydryl groups per average molecule; V. reoxidation of samples 

 containing about four sulfhydryl groups per average molecule.^ 



