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



that in some cases large effects on reactivity may also be the consequence of 

 neighbouring group participation, or the influence of electrostatic inter- 

 actions in the vicinity of the reacting groups. A further factor of import- 

 ance may be the ability of the protein molecule to absorb the reagent 

 specifically and thereby to facilitate the reaction of a proximally located 

 functional group by overcoming the usual entropy factor. Finally, the 

 introduction of modified amino acid residues into the protein fabric may 

 of itself induce variations in the reactivity of the as yet unsubstituted 

 residues, either by direct steric interference or by alteration of the tertiary 

 structure. In general, therefore, unless it becomes possible to locate a 

 number of the functional groups with unusual reactivity by direct reference 

 to the primary structure, there is little chance that observations on re- 

 activity alone can be of value in revealing specific aspects of the secondary 

 and tertiary structure. 



i-Fluoro-2,4-dinitrobenzene (FDNB) has a number of properties 

 desirable in a reagent that is to be used in studying the reactivity of func- 

 tional groups in a protein. It behaves in the manner of a typical reactive 

 alkyl halide* and is capable of reacting with unprotonated a-amino, 

 €-amino, and imidazole groups, as well as with thiol and phenolic hydroxyl 

 groups in the form of their conjugate bases. Introduction of the dini- 

 trophenyl group into the protein confers the characteristic spectral 

 properties of this chromophore to the new compound, a feature of value in 

 analysis, and excess reagent and dinitrophenol, formed from the reagent 

 by hydrolysis, are readily removed from the reaction mixture by extraction. 



The groups potentially sensitive to fluorodinitrobenzene in ribonu- 

 clease A comprise the single a-amino group of the lysine residue at the 

 amino-terminal end of the peptide chain, the lo e-amino groups of the 

 lysine residues at positions i, 7, 31, 37, 41, 61, 66, 91, 98, and 104, the 

 imidazole groups of the four histidine residues at positions 16, 48, 105, 

 and 119, and the phenolic hydroxyl groups of the six tyrosine residues at 

 positions 25, 73, 76, 92, 97, and 115. Under the conditions examined thus 

 far, fluorodinitrobenzene does not readily form sulphonium derivatives 

 with the thioether function of the methionine residues, nor is reaction 

 with the hydroxyl functions of the serine and threonine residues in 

 ribonuclease A appreciable. 



Analytical 



In the present studies the extent of substitution on dinitrophenylation 

 of the enzyme has been followed by quantitative amino acid analysis with 

 the method of Moore et al. [3] in conjunction with an automatic analyzer 



* Because of this similarity, we have elected to call substitution reactions 

 involving the introduction of the dinitrophenyl group alkylations, even though it 

 would be more accurate to use the term arvlation. 



