232 C. B. ANFINSEN [13 



not yet been achieved. However, it is of interest that the active, partially 

 reduced molecules, free of detectable amounts of native enzyme, are much 

 more susceptible to proteolytic digestion (Fig. 7) than the native protein 

 and that enzyme activity persists even after considerable further degrada- 

 tion with subtiHsin.^^ This latter observation is promising as regards the 

 production of a relatively small, catalytically active, 'core'. 



SH 



Native 

 



(-) 

 Reduced 



3.8 



5.1 



8.0 



Re-oxidized 

 3.4 



(Origin) 



■^Z2zm 





SZ 



Activity 100 



46 



21 

 (+) 



Fig. 6. Drawing of the disulfide-positive bands of the electrophoretic patterns obtained 

 on subtilisin digests of native, reduced, and reoxidized ribonuclease. Same conditions as 

 in Fig. 1a. The sample which was reoxidized with molecular oxygen contained 8-0 SH 

 groups and was completely inactive before oxidation.^* 



SUBTILISIN, 8SH 



200 



Fig. 7. Digestion of ribonuclease, at various stages of reduction, with subtilisin and 

 tiypsin. The reduced derivatives were carboxymethylated, before digestion. Alkali uptake 

 was followed in the Coleman autotitrator at pH 8. Temperature, 37°, except where indi- 

 cated. Substrate concentration, 10 mg/ml. Proteolytic enzyme concentration, 1 % of this 

 level.^^ 



