DINITROPHENYLATION OF RIBONUCLEASE A 43 



of the type described by Spackman et al. [4]. It should be emphasized 

 that this approach is hmited in its appUcation, in the ideal case, to the 

 study of substitutions that lead to the formation of derivatives capable of 

 withstanding the customary acid hydrolysis necessary for the breakdown 

 of the protein to amino acids. Moreover, in using acid hydrolysis the 

 possibility must be recognized that the appearance of a stable derivative 

 on hydrolysis may in itself constitute an artifact reflecting the consequences 

 of an acid-catalyzed rearrangement in which migration of the dinitro- 

 phenyl group from the functional group of original substitution is in- 

 volved. When substitution takes place with the formation of derivatives 

 unstable to acid hydrolysis, two broad alternatives are possible: the 

 derivative may decompose by reversal of its formation and thereby re- 

 generate the amino acid, or decomposition may take place into other 

 products without regeneration of the amino acid. The first of these 

 alternatives makes it essential that the degree of substitution of the protein 

 be checked prior to hydrolysis by means of absorption measurements in 

 the ultra-violet; the second requires that the degree of substitution be 

 measured by difference analysis after hydrolysis. 



Substitution on the amino-terminal lysine residue in ribonuclease A 

 may result in the formation of three products : a-dinitrophenyl-lysine, 

 e-dinitrophenyl-lysine, and a,e-bis-dinitrophenyl-lysine. e-Dinitrophenyl- 

 lysine is an a-amino acid and reacts with ninhydrin under the conditions 

 that obtain in the automatic amino acid analyzer to give a blue product 

 in approximately the same yield as typical a-amino acids. In our hands it 

 has proved to be stable (loss less than 5 "o after 22 hours at 110° in constant 

 boiling HCl) to the usual conditions of hydrolysis routinely employed as 

 a preliminary to quantitative amino acid analysis. a-Dinitrophenyl-lysine 

 contains a free e-amino group with the characteristics towards ninhydrin 

 of the amino group in y-aminobutyric acid. The relatively greater basicity 

 of the amino group in y-aminobutyric acid causes the compound to be 

 more strongly retained than a-aminobutyric acid upon chromatography 

 over columns of sulphonated polystyrene resins. Similarly, a-dinitro- 

 phenyl-lysine is more strongly retained by these resins than the e-isomer. 

 The presence of a dinitrophenyl radical in both derivatives confers on them 

 an enhanced affinity for the matrix of polystyrene based ion-exchangers. 

 Thus, when a mixture of the common protein amino acids and e-dinitro- 

 phenyl-lysine is chromatographed on the 15 cm. column of Amberlite 

 IR-120, normally used for the separation of the basic amino acids on the 

 automatic analyzer, the dinitrophenyl derivative emerges 35 effluent ml. 

 after arginine. a-Dinitrophenyl-lysine is even more strongly retained and 

 is not eluted satisfactorily from the column under the same conditions. 

 a,e-Bis-dinitrophenyl-lysine does not react with ninhydrin and is so firmly 

 bound by Amberlite IR-120 that only drastic conditions will serve to 



