- A kinetic approach to characterize the electrostatic environments of thiol groups in proteins
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In this study, we synthesized a zwitterionic DTNB derivative, 5-(2- aminoethyl)-dithio-2-nitrobenzoate (ADNB), and characterized its reactions with several cationic, anionic, and neutral thiols. Reactions with ADNB, unlike those with DTNB, are relatively insensitive to electrostatic environments and ionic strengths. At relatively low ionic strength, rate ratios, k(ADNB)/k(DTNB), varied from 0.22 for reactions with low-molecular- weight cationic thiols to 3.0 for those with low-molecular-weight anionic thiols. A k(ADNB)/k(DTNB) ratio of ~200 for Cys-34 of BSA appears to reflect a very anionic environment. k(ADNB)/k(DTNB) ratios of ~6 and ~1, respectively, for canine and equine serum albumins, which have Glu-82 → Asp and Glu-82 → Ala substitutions suggest Glu-82 is the most important anionic residues affecting the reactivity of Cys-34 in BSA. k(ADNB)/k(DTNB) ratios appear to be useful for characterizing electrostatic environments of thiol groups in proteins.
- Zhang, Hao,Le, Min,Means, Gary E.
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p. 356 - 364
(2007/10/03)
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- Preparation and Characterization of a Cleavable Photoacivable Heterobifunctional Fluorescent Reagent
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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 ca. 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.
- Muramoto, Koji,Kamiya, Hisao
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p. 547 - 554
(2007/10/02)
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