13] STRUCTURE & ENZYMATIC ACTIVITY OF RÏBONUCLEASE 225 



o o 



/ / 



. . .C— NH— CH— C— NH. . 



O 



CHs 



I 

 S 



I 

 S 



I 



CHo 



Thioglycollate 

 pH 8, 25°C 



O 



O O 

 / / ICHoCOO- 

 C— NH— CH— C— NH. . . > 



I pH 8, 25°C 



CH2 



SH 



\ I \ 



. . . HN— C— CH— NH— C ... 



00 



/ / 



. . .C— NH— CH— C— NH. . . 



I < V 1 



CHo 



I 



S— CH2— coo- 



The quantitative character of the reduction and alkylation procedures has 

 been confirmed by titration of the reduced proteins with /?-chloromercuri- 

 benzoate and by the isolation and determination of A^-dinitrophenyl-^"- 

 carboxymethyl-L-cysteine from dinitrophenylated hydrolysates of the alkylated 

 derivatives. 2 Table 1 shows data comparing the determination of SH groups 

 by the two methods in ribonuclease at different stages of reduction. As can 



Table 1 



ANALYSIS FOR CYSTEINE IN RIBONUCLEASE AT VARIOUS 



STAGES OF REDUCTION, BY /7-CHLOROMERCURIBENZOATE 



(PCMB) TITRATION* AND BY ESTIMATION OF 



A^-DINITROPHENYL-5-CARBOXYMETHYL-CYSTEINE 



* Determined in dinitrophenylated hydrolysates of reduced, alkylated protein. Cor- 

 rected for losses due to hydrolysis and chromatography essentially as described by Levy.^ 

 Destruction of carboxymethylcysteine during acid hydrolysis is on the order of 5 % or 

 less (16 hours in constant-boiling HCl at 110°C.). 



Pps 



