Reversible fluorescence labeling of amino groups of protein using dansylaminomethylmaleic anhydride

The reversible fluorescence labeling of insulin, catalase and lysozyme has been demonstrated. As a derivatizing reagent, dansylaminomethylmaleic acid (DAM) has been used after investigating the precolumn and precapillary derivatization conditions. This reagent (DAM) reacts with the amino groups of proteins via its anhydride in the presence of a suitable dehydrating reagent, which then could be liberated under mild acidic conditions and the native proteins are regenerated. After the derivatization of insulin, catalase and lysozyme with DAM, no peaks of these native proteins were observed while several peaks of the derivatized proteins due to the multiple labeling were observed. However, after the regeneration, increasing amounts of the native proteins were observed as the regeneration period increased. For the lysozyme, the bacteriolytic activity of the enzyme decreased after the derivatization, and only 0.9% of the activity remained. The activity increases by the regeneration, and 95.6% of the bacteriolytic activity of the native enzyme was observed after a 48-h regeneration at pH 2.5 and 40 degrees C.