A new double-labelling procedure for determination of amino acid composition: Application to bacteriorhodopsin

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Abstract
A new double-labeling procedure for amino acid analysis which requires only routine chromatographic equipment is described. When 1-fluoro-2,4-dinitro[3H]benzene is reacted with a mixture of 14C-labeled amino acids followed by reaction with the same 14C-labeled amino acid mixture diluted with an unlabeled sample of amino acids, the 3H:14C ratio in the resulting 2,4-dinitrophenyl (DNP) amino acid derivatives of the diluted sample will be increased in proportion to the quantity of unlabeled amino acid in the diluted sample. This procedure gave reliable results when applied to the known proteins insulin and lysozyme. The procedure is most advantageous when applied to amino acids which are unstable during acid hydrolysis or present in low molar fractions. When the bacteriorhodopsin in Halobacterium cutirubrum was analyzed, this procedure showed the presence of one histidine residue and 4 tryptophan residues/mol protein but no cystine or cysteine. In general, the analyses obtained were consistent with those originally reported by Oesterhelt, D. and Stoeckenius, W.

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