DINITROPHENYLATIOX OF RIBONUCLEASE A 55 



thus reveals that modification of a lysine residue in 0-Tryp 9 must have 

 taken place as the only consequence of dinitrophenylation. There are two 

 lysine residues in peptide 0-Tryp 9. Since the peptide following 0-Tryp 9 

 in the amino acid sequence is 0-Tryp 5, had dinitrophenylation taken 

 place at the carboxyl-terminal lysine residue of peptide 0-Tryp 9, tryptic 

 hydrolysis at this residue would have been blocked and 0-Tryp 5 could 

 not have formed. Since peptide 0-Tryp 5 was present in the tryptic 

 hydrolysate, the dinitrophenylated lysine residue in 0-Tryp 9 must have 

 been the lysine residue near the amino-terminal end of this peptide. 

 Examination of the amino acid sequence [i] shows that this is the lysine 

 residue at position 41 along the chain. Experiments on the further charac- 

 terization of the inactive protein, and on the isolation of dinitrophenylated 

 peptide derivatives are now in progress. 



Conclusions 



These preliminarv studies have revealed that amino groups are the 

 most reactive functional groups in ribonuclease towards substitution by 

 dinitrofluorobenzene at pH 8. Two e-amino groups react faster than the 

 a-amino group of the lysine residue at the amino-terminal end of the pep- 

 tide chain. The substitution of one of the rapidly reacting e-amino groups 

 is accompanied bv the inactivation of the enzyme. This group is the 

 e-amino group of the lysine residue at position 41. 



The ease of substitution of the e-amino group of the lysine residue at 

 position 41 is of interest in relation to the primary structure of the protein. 

 One of the relativelv few unique features of the amino acid sequence is 

 the accumulation of basic amino acid residues in the region between 

 residues 31 and 41. As pointed out elsewhere [i], there are five residues 

 capable of conferring a positive charge in these 1 1 residues, and only one 

 carboxyl group. A further structural feature of significance is that lysine 

 residue 41 is preceded by half-cystine residue 40 and followed by proline 

 residue 42. The combined influence of charge repulsion at pH 8 among 

 the cationic centres and the influence of the adjoining cystine and proline 

 residues would be factors adversely affecting the stability of an a-helix 

 formed in this part of the molecule. An additional feature of note is that 

 half-cystine 40 is the first half-cystine of the II-VII disulphide bond [2]. 

 Examination of the primary structural formula will reveal that the II-VII 

 disulphide bond is a fulcrum about which any folding of the ribonuclease 

 molecule must hinge. The ease of substitution of lysine residue 41 may 

 therefore indeed reflect a greater degree of accessibility of this residue 

 because of less compact folding of the structure in this region. 



The inhibition of dinitrophenylation at lysine residue 41 by cytidylate 

 and pyrophosphate is extremely effective even when only one equivalent 



