DINITROPHENYLATION OF RIBONUCLEASE A 5 1 



the action of fluorodinitrobenzene. Their detection by kinetic methods 

 would demand a greater degree of refinement in the analytical work than 

 has been attained thus far. On the other hand, reaction of fluorodinitro- 

 benzene at these more slowly reacting groups may not be inhibited as 

 effectively by the agents already discussed, and their detection might 

 therefore be facilitated by the study of the consequences of dinitrophenyl- 

 ation in the presence of the competitive inhibitor of the enzyme. 



Isolation of inactivated mono-e-dinitrophenylaminoribonuclease A 



In an experiment similar to that described in Table I, aliquots of the 

 reaction mixture of ribonuclease A (acetate) and fluorodinitrobenzene at 

 pH 8 were chromatographed on an analytical scale on columns of the 

 sodium form of IRC-50 equilibrated with 0-2 m sodium phosphate buffer 

 at pH 6-47 [11]. The chromatograms sho\\ed that the reaction mixture 

 rapidly becomes complex. At higher degrees of substitution, the products 

 present could not be eluted satisfactorily from the resin. 



When the products formed from 100 mg. of ribonuclease A (acetate) 

 inactivated to the extent of 20'',, were chromatographed on a larger scale 

 the result shown in Fig. 3 was obtained. The elution diagram shows a 

 simultaneous plot of the absorbance, determined at 360 m^i, and of the 

 ninhydrin colour value developed by aliquots of the eflluent fractions. The 

 values for the absorbance at 360 m^ and for the ninhydrin colour value 

 are normalized at the maximum of the elution peak at 350 eflluent ml. As 

 expected, the peak at 43:; eflluent ml. was found to represent unchanged 

 ribonuclease A. The trailing shoulder of the peak at 350 ml. exhibits an 

 esentially constant ratio of the ninhydrin colour value to absorbance at 

 360 m/i, suggesting the presence of material of a constant degree of sub- 

 stitution. Aliquots of the fractions in this portion of the chromatogram 

 when subjected to ribonuclease activity determinations were found to be 

 devoid of activity. As little as o-2"o of the original specific activity could 

 have been detected by the procedure used. Partly overlapping the peak 

 at 350 ml. on the chromatogram is a smaller peak at 325 ml., exhibiting a 

 variable ninhydrin colour value to absorbance at 360 m/x ratio. Material 

 corresponding to the fractions in this peak was enzymically active, as was 

 the protein in the fractions corresponding to the relatively unretarded 

 peaks, with varying ninhydrin colour value to absorbance at 360 m^ ratios, 

 between 250 and 300 efiluent ml. The latter evidently represent a mixture 

 of different partly substituted proteins with ribonuclease activity. 



A cut was made of the yellow protein represented by the trailing 

 shoulder of the peak at 350 ml., and the resulting solution was freed of 

 buffer salts by gel filtration over a column of Sephadex G-25 [12]. On 

 rechromatography under identical conditions of elution the inactive 



