DINITROPHENYLATION OF RIBONUCLEASE A 45 



Dinitrophenylation of ribonuclease A 



In view of the strong affinity of ribonuclease for polyvalent anions (cf. 

 discussion bv Stein [5]) and the possibility that interaction with such 

 ions might modify the reactivity of some of the histidine and lysine side 

 chains, the ribonuclease A used in the present studies was subjected to a 

 preliminary ion-exchange step in which conversion to the acetate of the 

 protein was achieved. 



The reaction between the protein and fluorodinitrobenzene was carried 

 out in aqueous solution at a constant pH, maintained with a pH-stat. A 

 large excess of reagent was used, and the solution was kept saturated with 

 reagent by vigorous stirring. As pointed out by Levy [6], under these 

 conditions the kinetics of the reaction with a particular functional group 

 become pseudo first-order in nature. The reaction may be stopped by 

 acidification to a pH of about 2, whereupon excess reagent and dinitro- 

 phenol may be removed by extraction. 



Ribonuclease A can be completely dinitrophenylated. There are no 

 residues inaccessible to fluorodinitrobenzene, though it is evident that 

 wide variations in reactivity are to be found among the groups under- 

 going substitution. At pH 10, and at 40^, for example, approximately 30 

 hours are required for complete dinitrophenylation. After this time, some 

 0*3 residues of lysine remain unsubstituted, while free histidine and 

 tyrosine are absent from hydrolysates of the modified protein. After 15 

 hours under the same conditions, the equivalent of one lysine residue 

 remains unsubstituted, while reaction with histidine and tyrosine is com- 

 plete. It is therefore clear that a number (possibly one or two) of the 

 e-amino groups in the protein have decreased reactivity even at pH 10. 

 The propertv is probablv dependent on some feature of the primary struc- 

 ture, because performic acid-oxidized ribonuclease A exhibits similar 

 behaviour on dinitrophenylation. 



It is noteworthy that completely dinitrophenylated ribonuclease A is a 

 soluble protein in the pH range from 2 to 10. This rather unusual property 

 is of value in handling the partly substituted derivatives of ribonuclease 

 A, for it permits the facile separation of excess fluorodinitrobenzene from 

 the protein. 



Of more immediate interest in the present contribution are the residues 

 in ribonuclease A that are most reactive towards substitution by fluoro- 

 dinitrobenzene. At pH 8 analysis indicates that certain e-amino groups 

 are substituted more rapidly than any other functional groups, including 

 the a-amino group. This will be apparent from Table I, in which are given 

 the results obtained upon quantitative amino acid analysis of aliquots 

 removed after successive time intervals from a reaction mixture at pH 

 8 and at 15 . The values for all the residues in ribonuclease are shown, and 



