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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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Published in: | Analytical chemistry (Washington) 2003-05, Vol.75 (10), p.2512-2516 |
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Main Authors: | , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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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. |
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ISSN: | 0003-2700 1520-6882 |
DOI: | 10.1021/ac026279e |