Synthesis and characterization of a novel reagent containing dansyl group, which specifically alkylates sulfhydryl group: an example of application for protein chemistry |
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Authors: | Hasegawa Go Kikuchi Masashi Kobayashi Yuichi Saito Yuji |
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Affiliation: | Department of Biological Sciences, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan. |
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Abstract: | We have synthesized a novel reagent containing dansyl group, iodoacethyl dansylcadaverine (IADC), which specifically alkylates sulfhydryl groups. The carboxyl group of iodoacetic acid was activated with dicyclohexylcarbodiimide and was condensed with amino group of dansylcadaverine. Purity and chemical structure of IADC was confirmed with mass spectrometry (MS) and NMR. IADC alkylated GSH but not GSSG, which was confirmed by MS. The reactivity of IADC with proteins was also investigated with Western blotting using anti-dansyl antibody. IADC reacted only with sulfhydryl-containing proteins. The specificity of the interaction of IADC with sulfhydryl groups in proteins was confirmed by adding excessive amount of a well-known sulfhydryl-specific reagent, 5, 5'-dithiobis(2-nitrobenzoic acid), which led to a complete inhibition. To show the usefulness of IADC, the cysteines in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from chicken muscle were modified with this reagent, and GAPDH was then digested by lysyl endopeptidase. The peptides generated from digestion of IADC-incorporated GAPDH were applied to an anti-dansyl immunoaffinity column. The peptide fragments bound and eluted from the column were separated by HPLC, and the amino acid sequence of each peptide was analyzed, and peptide was identified as the one containing a Cys residue(s). These data showed that IADC is a useful reagent to specifically identify the positions of a Cys residue(s) in proteins. |
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Keywords: | IADC, iodoacethyl dansylcadaverine GAPDH, glyceraldehyde-3-phosphate dehydrogenase GSH, glutathione of reduced form GSSG, glutathione of oxidized form IAA, iodoacetic acid DCC, N,N′-dicyclohexylcarbodiimide Lys-C, lysyl endopeptidase IAM, iodoacetamide MDC, monodansyl cadaverine DTNB, 5, 5′-dithiobis(2-nitrobenzoic acid) rd-RNase, reduced RNase rd-lysozyme, reduced lysozyme rd-BSA, reduced BSA |
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