Colorimetry of dehydroalanine residues preserved as 'lost side chains' in thyroglobulin.

We developed a new assay method for dehydroalanine residues in thyroglobulin, which had been proposed to be the 'lost side chains' during thyroid hormonogenesis. Thyroglobulin preparations were labeled with 4-aminothiophenol at 30 degrees C for 10 days. Under the conditions, the reagent reacted only with dehydroalanine and cysteine residues. The 4-aminothiophenol bound to cysteine was eliminated by reductive cleavage. The 4-aminothiophenol-labeled dehydroalanine (4-aminophenylcysteine) residues were liberated by acidic hydrolysis, converted to a colored derivative by the Bratton-Marshall reaction and quantified colorimetrically. The number of dehydroalanine residues was the same as that of hormone residues in each thyroglobulin preparation. The results indicate that when one hormone residue is produced by the coupling of two iodotyrosine residues, the 'lost side chain' is preserved as one dehydroalanine residue in the thyroglobulin molecule.