DINITROPHENYLATION OF RIBONUCLEASE A 49 



detected. At present, however, it is evident that the presence of inhibitor 

 serves to slow the reaction down at relatively few of the available functional 

 groups on the protein molecule. 



Examination of the values in Table I suggested that the inactivation 

 reaction was primarily due to substitution at an e-amino group. Ray and 

 Koshland have recently developed a treatment for dealing with the results 



Fig. I. Kinetics of inactivation of ribonuclease A by dinitrophenylation. An 

 initial lag phase in the reaction is not shown. The conditions of dinitrophenylation 

 were the same as those described in Table I. The points represent the average of 

 two determinations at different concentrations of the remaining ribonuclease 

 activity. The procedure of Gundlach et ol. [8] was used with cyclic cytidylic acid 

 as the substrate. 



obtained by kinetic analysis of modification reactions with proteins [lo]. 

 With this approach, and on the assumption that during the initial stages 

 of dinitrophenylation (up to 40 minutes in Table I) the drop in the total 

 lysine value is due exclusively to reaction of fluorodinitrobenzene at but 

 two e-amino groups, a pseudo first-order reaction rate constant for these 

 two groups of o- DIG min.~^ was calculated. Determination of the remaining 

 ribonuclease activity in the reaction products during the same experiment 



