Abstract
The fluorescent Kashland reagent, 2-carboxy-1-hydroxy-4-naphthylmethyldimethylsulfonium chloride (FKR), has three different functions : a) a phenolic hydroxy group ortho to carboxy group to chelate metal ions; b) a naphthyl moiety to produce fluorescence; and c) a Koshland reagent-type sulfonium chloride group to modify tryptophyl residues selectively in a peptide chain. Three procedures, methods I, II and III, using this reagent for the selective isolation of tryptophan-containing peptides from a protein digest were investigated. We selected lysozyme and carboxymethylated lysozyme as model proteins. In method I, lysozyme was labeled with FKR prior to the enzyme digestion, and the labeled protein was purified by gel permeation chromatography to remove the by-product formed from the reagent, then digested with protease. In method II, the protein (carboxymethylated lysozyme) was similarly labeled with the reagent, and the labeled protein was recovered as a precipitate by adding ethanol, leaving the excess reagent in the supernatant. The labeled protein was digested with protease. In method III, the protein was digested by protease before treatment with the reagent, and the by-product was removed by extraction with benzene. When the digests obtained by these procedures were applied to a Chelating Sepharose 6B column (Fe3+ or Al3+ form), FKR-labeled tryptophan-containing peptides were selectively adsorbed on the column owing to its chelating ability and could be eluted with 50 mM ethylenediaminetetraacetic acid. The final purification of these peptides was performed by reversed-phase high performance liquid chromatography on an octadecylsilane column in all cases.
