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Spectrophotometric Tool for the Determination of the Total Carboxylate Content in Proteins; Molar Extinction Coefficient of the Enol Ester from Woodward's Reagent K Reacted with Protein Carboxylates

A number of relevant properties of Woodward's reagent K have been determined, such as the stability of the reactant and the optimal reaction conditions of the reactant with protein carboxylates. A Woodward's reagent K stock solution was stable at 4 °C for prolonged time, whereas upon stora...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2003-05, Vol.75 (10), p.2512-2516
Main Authors: Kosters, Hans A, de Jongh, Harmen H. J
Format: Article
Language:English
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Summary:A number of relevant properties of Woodward's reagent K have been determined, such as the stability of the reactant and the optimal reaction conditions of the reactant with protein carboxylates. A Woodward's reagent K stock solution was stable at 4 °C for prolonged time, whereas upon storage at 22 °C, almost 20% of the reactive compound was lost within 1 week. The pH-dependency of the spontaneous degradation reaction of Woodward's reagent K was studied and was shown to be base-mediated. A molar extinction coefficient of 3150 M-1 cm-1 at 269 nm for the enol ester resulting from the reaction between Woodward's reagent K and the protein carboxylates was established using the conditions laid out in this work. This value was validated using a variety of proteins that were modified by Woodward's reagent K. In addition, upon methylation of the carboxylates of a single protein, ovalbumin in this case, the degree of modification could be determined accurately and was confirmed by cation exchange chromatography elution profiles.
ISSN:0003-2700
1520-6882
DOI:10.1021/ac026279e