52 



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



dinitrophenylated ribonuclease A was eluted as a symmetrical peak at the 

 same position. A mixture of ribonuclease A and the inactivated protein 

 separated in the manner to be expected from Fig. 3. When the inactive 

 protein was rechromatographed on IRC-50 in 0-2 m sodium phosphate 

 buffer at pH 6-02 [13] a single, symmetrical peak was again obtained on 



EFFLUENT VOLUME ml 



Fig. 3. Chromatography of the reaction mixture from ribonuclease A (acetate) 

 and dinitrofiuorobenzene at pH 8 and 15 after the attainment of 20°,, inactivation. 

 Conditions for the reaction were the same as described in Table I. The IRC-50 

 column measured 37 x 330 mm. and was equilibrated with o • 2 M sodium phosphate 

 buffer at pH 6 -47. The rate of elution was 15 ml. /hour and the effluent was collected 

 in 5 ml. fractions. The absorbance of the effluent fractions was measured at 360 m/i 

 with a Beckman DU spectrophotometer. Aliquots of the fractions were also sub- 

 jected to ninhydrin analysis (cf. [11]), and to ribonuclease activity determinations 

 by the procedure described in Fig. i. 



the elution curve with a maximum at 3 -6 times the effluent volume of the 

 maximum observed at pH 6-47. 



On quantitative amino acid analysis the protein from the 350 ml. 

 elution peak in Fig. 3 gave the results shown in Table II. For comparison, 

 the results obtained on analysis of the ribonuclease A (acetate) used in 

 this experiment are also included. The values for all the residues agree 

 satisfactorily with the exception of those for lysine. The analysis makes it 



