50 



C. H. W. HIRS, M. HALMANN, J. H. KYCIA 



permitted the evaluation of a pseudo first-order rate constant of o-oio 

 min.~^ (on the basis of decimal logarithms) for the inactivation reaction. 

 The results are represented graphically in Fig. 2, in which the difference 

 in time scale for the lysine and activity values are occasioned by the dif- 

 ference of 2 minutes in the lag phases observed for the dinitrophenylation 

 and inactivation reactions. The agreement in the values is probably to 

 some extent fortuitous in view of the usual errors that obtain in the 



10 



J I L 



J \ L 



80 



10 20 40 60 



MINUTES 



Fig. 2. Kinetics of inactivation of ribonuclease A by dinitrophenylation. 

 Conditions were the same as described in Table I. For the significance of the 

 lysine values, see the text. 



determination of lysine and the problems involved in removing excess 

 reagent before undertaking the measurement of ribonuclease activity. The 

 values for the constant nevertheless furnish quantitative evidence that the 

 substitution of a single e-amino group is capable of inactivating the enzyme. 

 The powerful inhibition effected by the nucleotides and pyrophosphate 

 ion suggest that the e-amino group in question is located at either the 

 binding or catalytic site of the molecule. 



It is likely that other groups essential to the activity of the enzyme are 

 among those that become substituted much more slowly at pH 8 through 



