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



probably associated mainly with the process of saturating the solution 

 with fluorodinitrobenzene, the inactivation follows pseudo first-order 

 kinetics in the range from zero to about 95% inactivation. Representative 

 kinetic runs at three temperatures are shown in Fig. i. Ribonuclease activity 

 was measured with the aid of a pH-stat by the method of Gundlach et al. 

 [8] using cyclic cytidylic acid as the substrate. In the initial phases of the 

 work measurements of depolymerase activity were also effected. The 

 spectrophotometric method of Kunitz [9] with purified yeast ribonucleic 

 acid as substrate was employed with modifications to permit the use of a 

 Gary recording spectrophotometer. The results obtained with this method, 

 though less precise than those obtained with the nucleotide substrate, 

 gave the same kinetic constants. 



Determinations of the pseudo first-order constant at 15° gave values 

 ranging from o-oio to 0-020 min."^ (based on decimal logarithms). The 

 same sample of ribonuclease A (acetate) gave values that agreed within 

 10%, but different samples of ribonuclease A (acetate) gave significantly 

 different values for "k". A possible explanation for this variation became 

 apparent subsequently when it was found that the inactivation of ribonu- 

 clease A by dinitrophenylation was strongly inhibited by pyrophosphate 

 ion. A quantity of pyrophosphate equivalent to that of ribonuclease A in 

 an experiment of the type shown in Fig. i was sufficient to decrease the 

 rate of inactivation one-fifth. It is possible that the reaction would also be 

 inhibited by other polyvalent anions, such as phosphate and sulphate, 

 just as the inactivation of the enzyme by carboxymethylation on histidine 

 is inhibited by such ions [5]. Because of the methods of preparation different 

 samples of ribonuclease A (acetate) could well be contaminated to varying 

 extents by orthophosphate ions. 



The inactivation reaction at pH 8 is also inhibited by adenylate, 

 cytidylate, and uridylate. Inhibition by the pyrimidine nucleotides, which 

 are well known competitive inhibitors of ribonuclease, has been found to 

 be particularly effective. Cytidylate at equal concentration to ribonuclease 

 in an experiment of the kind shown in Fig. i caused the rate of inactivation 

 to be slowed to approximately one-quarter of the rate in the absence of 

 the inhibitor. At a ratio of 10 equivalents of cytidylate to i of ribonuclease 

 A inhibition was essentially complete. Adenylate was approximately half 

 as effective as cytidylate at the same concentration. 



While the inactivation of the enzyme by dinitrophenylation was in- 

 hibited by these anions, the over-all reaction with fluorodinitrobenzene, 

 as measured by alkali uptake in the pH-stat, was not detectably slowed 

 down. Thus far a detailed analysis of the kinetics of the reaction as 

 measured with the pH-stat has not been attempted, but it is likely that, 

 with improved technique and working on a larger scale, a difference of 

 rate in the initial stages in the presence and absence of inhibitor will be 



