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



remove it from the resin. Both a-dinitrophenyl-lysine and a,e-bis-dinitro- 

 phenyl-lysine undergo decomposition during acid hydrolysis prior to 

 amino acid analysis to varying extents up to 30% in 22 hours, but lysine 

 is not regenerated in the process. 



The histidine residues in ribonuclease A react with fluorodinitro- 

 benzene to form /w/V/rt;?o/f'-dinitrophenyl derivatives. The tautomerism of 

 the imidazole nucleus makes it possible for two dinitrophenyl derivatives 

 to be formed at each histidine residue. On hydrolysis in acid these deriva- 

 tives appear as the /w-dinitrophenylhistidines, unstable compounds that 

 break down into an as yet unidentified substance, with the properties of 

 an amino acid, that persists as the stable end-product of the acid hydrolysis. 

 Free histidine is not regenerated. The unidentified substance is eluted 

 more rapidly than histidine on the 15 cm. column used for the analysis 

 of the basic amino acids with the automatic analyzer, at a position 12 

 effluent ml. before lysine. 



Tyrosine residues in ribonuclease A become modified at the phenolic 

 hydroxyl group by fluorodinitrobenzene. The resulting tyrosine ether 

 residues may be measured as o-dinitrophenyltyrosine subsequent to 

 hydrolysis. Under the conditions of acid hydrolysis used in the present 

 work, o-dinitrophenyltyrosine has proved to be a stable compound. It 

 is bound strongly by Amberlite IR-120 and is not eluted from the resin 

 under the conditions used in the automatic analyzer. 



These observations on the dinitrophenyl derivatives of lysine, histidine, 

 and tyrosine have permitted the use of difference analysis to estimate the 

 extent of substitution of the ribonuclease A molecule on dinitrophenylation. 



Completely dinitrophenylated ribonuclease A on hydrolysis yields 

 nine equivalents of e-dinitrophenyl-lysine and one equivalent of a,e-bis- 

 dinitrophenyl-lysine per molecule. At intermediate stages of substitution, 

 blocking of the a-amino group of lysine at the amino terminus of the peptide 

 chain in ribonuclease A is revealed by the inequality between ten equiva- 

 lents (the total lysine available) and the sum of the equivalents of unreacted 

 lysine and e-dinitrophenyl-lysine. 



With histidine an independent check on the values obtained by dif- 

 ference is provided by the quantity of the unidentified degradation 

 product of /w-dinitrophenylhistidine formed during acid hydrolysis. 

 Free tyrosine, unlike histidine and lysine, undergoes destruction during 

 the acid hydrolysis normally used for amino acid analysis to the extent 

 of 5 to 8 %. Difference analysis as a measure of substitution of the six 

 tyrosine residues in ribonuclease A is therefore rendered uncertain to the 

 extent of the slight variations normally observed in the destruction of this 

 amino acid. For more reliable values, particularly at low degrees of sub- 

 stitution, it would be preferable to determine the o-dinitrophenyltyrosine 

 in the hydrolysates directly. 



