Preparation and Characterization of a Cleavable Photoactivable Heterobifunctional Fluorescent Reagent

Koji Muramoto, Hisao Kamiya

    Research output: Contribution to journalArticlepeer-review

    8 Citations (Scopus)


    A cleavable photoactivable heterobifunctional reagent, the N-hydroxysuccinimide ester of 1-azido-5-naphthalene sulfonyl S-carboxymethylthiocysteamine (NHS-ANS-CTC), was synthesized and characterized. The reagent was applicable to the group-directed modification of protein ligands, such as an invertebrate lectin and a trypsin inhibitor. The modified ligands bound to rabbit erythrocyte ghosts and trypsin, respectively. Upon exposure to ultraviolet light, the modified ligands reacted with their binding proteins to form cross-linked fluorescent products. The cross-linked fluorescent complexes were readily cleaved by reducing the disulfide bond of the reagent, leaving the fluorescent probe on the binding proteins. The photolabeled binding proteins were analyzed by SDS-polyacrylamide gel electrophoresis. The fluorescence intensity of the fluorescent probe was enhanced by 4 ∼ 8 times to improve sensitivity when the SDS-gel was dehydrated with methanol. This phenomenon was also observed with the proteins labeled with 1-dimethylamino-5-naphthalene sulfonyl chloride.

    Original languageEnglish
    Pages (from-to)547-554
    Number of pages8
    JournalAgricultural and Biological Chemistry
    Issue number2
    Publication statusPublished - 1988 Jan 1

    ASJC Scopus subject areas

    • Biochemistry, Genetics and Molecular Biology(all)
    • Agricultural and Biological Sciences(all)


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