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Fluorometric Determination of Thiol and Disulfide Groups in Protein Using N-(9-Acridinyl)maleimide

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Abstract

A sensitive fluorometric method has been developed to determine the thiol and disulfide groups in proteins using N-(9-acridinyl)maleimide as a fluorogenic reagent. Proteins are hydrolyzed with subtilopeptidase (Carlsberg type) in order to eliminate the steric hindrance. Then, thiols are determined by the reaction of A/-(9-acridinyl)maleimide with the hydrolysate. Disulfide can be determined using the same hydrolysate after the derivatization into thiols by electrolytical reduction. The fluorescence intensities have shown linear relations between the thiol and disulfide contents at nmol/ml levels in the model proteins. The sensitivity is 1000-times higher than conventional colorimetry with 5,5’-dithiobis(2-nitrobenzoate).

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Authors and Affiliations

  1. Laboratory of Food Hygiene, Tohoku Fukushi University, Kunimi, Aoba, Sendai, 981, Japan

    Eiko Hatakeyama, Norikazu Matsumoto & Takeo Ochi

  2. Laboratory of Wholesomeness Evaluation, National Food Research Institute, Kannondai, Tsukuba, 305, Japan

    Tateo Suzuki

  3. Laboratory of Analytical Chemistry, Faculty of Agriculture, Tohoku University, Tsutsumidori-Amamiyamachi, Aoba, Sendai, 980, Japan

    Hiroshi Ohrui & Hiroshi Meguro

Authors
  1. Eiko Hatakeyama
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  2. Norikazu Matsumoto
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  3. Takeo Ochi
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  4. Tateo Suzuki
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  5. Hiroshi Ohrui
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  6. Hiroshi Meguro
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Hatakeyama, E., Matsumoto, N., Ochi, T. et al. Fluorometric Determination of Thiol and Disulfide Groups in Protein Using N-(9-Acridinyl)maleimide. ANAL. SCI. 5, 657–661 (1989). https://doi.org/10.2116/analsci.5.657

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  • DOI: https://doi.org/10.2116/analsci.5.657

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