230 C. B. ANFINSEN [13 



Asp(NH2)-CyS03-Arg, rather than CyS03-Asp(NH2)-Arg. The former has 

 been deduced by Dr Hirs using the Edman degradative method, while 

 the latter is from our less rigorous studies using partial acid hydrolysis.) The 

 geometrical restrictions introduced into the ribonuclease molecule by the 

 disulfide bridges are severe and suggest that the marked changes ^^ that 



(Thr 



Q©©©©©©©©@, 



iLys) 



^^ö©@@©©©©©(Sk 



§ e0©©©@ 



© @®@@©©©©® ' 



ASP) 



Glyl^CyO 



ii^@@@(Q(sèr)@(S 



Fig. 3. Schematic diagram of ribonuclease structure. An represents asparagine; Gn 

 glutamine. The location of amide groups is possible from the work of Hirs, Moore, and 

 Stein. (See p. 218.) 



occur when the protein is dissolved under 'denaturing' conditions such as 

 8m urea are due to the unfolding of the uncrosslinked 'tails' and perhaps 

 to partial expansion of coiled regions between S-S bridges. 



I would like now to outline, in a brief way, the results of studies on the 

 relationships between the structure and function of ribonuclease. 



1. REDUCTIVE CLEAVAGE OF DISULFIDE BRIDGES^ 



As discussed above, thioglycollic acid causes a rapid reduction of disulfide 

 bridges in ribonuclease in the presence of 8m urea at pH values in the neigh- 

 borhood of 8. In the absence of urea, however, only partial reduction occurs, 

 and in a way which suggests that two of the four bridges in the native pro- 

 tein are particularly susceptible (Fig. 4). During the reaction the activity of 

 the enzyme decreases in a manner (Fig. 5) which indicates that certainly 

 one, and perhaps two, of the bridges are not essential for catalysis. »S-Carb- 

 oxymethylation of the SH groups does not affect the enzyme activity of the 



