226 C. B. ANFINSEN [13 



be seen here, the chromatographic separation of A^-dinitrophenyl-^-carboxy- 

 methylcysteine and its subsequent elution and estimation compares well with 

 the spectrophotometric method. Table 2 deals with similar experiments on 



Table 2 



ESTIMATION OF CYSTEINE, ASPARTIC ACID, 

 AND GLUTAMIC ACID CONTENT OF FULLY 

 REDUCED, CARBOXYMETHYLATED LYSOZYME 



The reduced, alkylated protein was hydrolyzed at 110° for 16 hours in 6n-HC1. The 

 hydrolysate was dinitrophenylated^ and, following chromatography according to Levy,^ 

 the spot containing the DNP derivatives of glutamic acid, aspartic acid, and S-carboxy- 

 methyl-cysteine was excized. The mixture of derivatives was separated in the tertiary amyl 

 alcohol system of Blackburn and Lowther,^ running the chromatograms in the descend- 

 ing direction for 48 hours. 



lysozyme and indicates that the application of the reductive and alkylating 

 techniques to this tryptophan-rich protein furnishes a convenient method 

 both for opening the cross-linked chain and for the subsequent determina- 

 tion of the cystine content of the protein. 



It has also been found that, under the conditions of reduction and alkyla- 

 tion employed, histidine, tryptophan, and tyrosine residues in the polypeptide 

 chain are unchanged. Michaelis and Schubert, in 1936,'^ demonstrated that 

 only SH groups are alkylated by haloacetic acids under sufficiently mild 

 conditions (room temperature and pH values in the neighbourhood of 8). 

 It has recently been reported by Korman and Clarke that more vigorous 

 conditions (40°, pH 9, 4-5 hours) are required for the alkylation of free 

 amino groups and that the ring nitrogen atoms and the phenolic hydroxyl 

 group of tyrosine are even more resistant to reaction.^ We have further 

 examined these possible side reactions under the conditions employed by us 

 (room temperature, pH 8, 2 hours) and find that less than 5% of the histidine 

 and tyrosine residues and free amino groups become carboxymethylated 

 under conditions where reaction of SH groups is complete, 



* The 'best' values as mentioned by Thompson.^ 



t The value for glutamic acid is undecided. Thompson's value of 7, deduced from 

 sequence studies, is probably high. Lewis et al.^^ give 4, as do Dose and Caputo,^^ and 

 Thompson^ gives 5-1 on the basis of amino acid analysis. 



X Calculated assuming the presence of 20 residues of aspartic acid in the protein. 



§ A^-dinitrophenyl-5-carboxymethylcysteine. 



