Fluorescent Koshland Reagent with Chelating Ability : Application to Selective Isolation of Tryptophan-Containing Peptide Fragments from Enzymatic Protein Digest(Analytical,Chemical)
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概要
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The fluorescent Koshland 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 carboxy-methylated 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 (Fe^<3+> or Al^<3+> 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.
- 公益社団法人日本薬学会の論文
- 1987-09-25
著者
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NAKAMURA Hiroshi
Faculty of Science and Technology, Ryukoku University
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Nakajima Terumi
Faculty Of Pharmaceutical Sciences University Of Tokyo
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Nakajima Terumi
Faculty Of Pharamaceutical Sciences The University Of Tokyo
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Nakamura Hiroshi
Faculty Of Pharmaceutical Sciences University Of Tokyo
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Nakamura Hiroshi
Faculty Of Pharmaceutical Sciences Science University Of Tokyo
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HAGIWARA KEN'ICHI
Faculty of Pharmaceutical Sciences, University of Tokyo
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