Studies on the determination of the sequence of amino acids in peptides and proteins. 2. The separation of the methyl esters of N-2:4-dinitrophenyl derivatives of amino acids by adsorption chromatography and the free amino groups of insulin

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Abstract
Methyl esters of N-2, 4-dinitrophenyl (DNP)derivatives of a number of amino acids were prepared. Separation of mixtures of these derivatives by adsorption chromatography on alumina columns was investigated. Stability to acid hydrolysis of some DNP-amino acids was investigated by 2 different methods. The new method of separation of mixtures of DNP-amino acids (by adsorption chromatography of their methyl esters) was applied to the re-investigation of the free amino groups of insulin. Assuming a molecular wt. of 12,000 it is shown that 2 free amino groups are on phenylalanine residues, 2 or 3 on glycine residues, and 2 represent the epsilon-amino group of lysine. In addition, a new, unidentified substance bearing free amino groups was found. It is suggested that owing to the difficulty of an exact assessment of the magnitude of correction factors for the decomposition of DNP-amino acids during protein hydrolysis, it must be considered as quite uncertain how far the calculated values for the amt. of N-terminal amino acid residues really correspond to the amt. actually contained in insulin.