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



in each case represent the resuhs of single determinations. The agreement 

 shown for the residues not affected by dinitrophenylation will serve to 

 give an impression of the degree of reproducibility attainable by this 



approach. 



TABLE I 



The Dinitrophenylation of Ribonuclease A at pH 8-o, 15° 



An aqueous 0-7 x lo"^ M solution of ribonuclease A (acetate) at pH 8-o and 

 at 15° was stirred vigorously with 20 times the quantity of i-fluoro-2,4-dinitro- 

 benzene required for the substitution of all the potentially available functional 

 groups in the protein. The initial volume was 13-6 ml. and the pH was maintained 

 at a value of 8-o±o- 1 with 0-2 N NaOH added with the aid of a pH-stat. Before 

 addition of the reagent an aliquot of the protein solution was removed, acidified 

 to pH I -8 with 006 N HCl, and the solution extracted 6 times with 20 volumes of 

 benzene. The protein was subjected to hydrolysis with constant boiling HCl in 

 an evacuated, sealed tube for 22 hours at iio\ after which the hydrolysate was 

 concentrated to remove excess acid, and the amino acid composition of the sample 

 determined by the method of Spackman et al. [4]. In a similar manner, aliquots of 

 the reaction mixture following the addition of FDNB were removed at successive 

 intervals of time, and the reaction in each case terminated by the addition of acid 

 to pH I -8. Thereafter, excess FDNB was removed by extraction, and the protein 

 subjected to quantitative amino acid analysis by the same procedure. The results 

 are expressed in terms of molar ratios of the constituent amino acids. Other aliquots 

 of the acidified, extracted reaction mixture were kept for determinations of 

 ribonuclease activity. 



♦ Incompletely liberated after hydrolysis for 22 hours, 

 t Correction for destruction on hydrolysis not applied. 



Examination of Table I reveals that the only values altering signifi- 

 cantly with time are those for lysine, histidine, and tyrosine. Methionine 

 recovery was poor throughout, but the values for the sum of free methio- 

 nine and the methionine sulpho.vides remained satisfactorily constant. 



