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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

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Author: Kosters, H.A. · Jongh, H.H.J.de
Type:article
Date:2003
Institution: TNO Voeding
Source:Analytical Chemistry, 10, 75, 2512-2516
Identifier: 237087
doi: doi:10.1021/ac026279e
Keywords: Nutrition · Food technology · Chemical modification · Chromatographic analysis · Degradation · Esters · pH effects · Spectrophotometry · Molar extinction · Proteins · chemical analysis · degradation · ion exchange chromatography · methylation · molecular stability · pH · protein modification · protein stability · reaction analysis · spectrophotometry · temperature dependence · validation process · Animals · Carboxylic Acids · Chickens · Esters · Isoxazoles · Kinetics · Ovalbumin · Proteins · Spectrophotometry, Ultraviolet

Abstract

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 basemediated. 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. Chemicals/CAS: ovalbumin, 77466-29-6; Carboxylic Acids; Esters; Isoxazoles; N-ethyl-5-phenylisoxazolium-3'-sulfonate, 4156-16-5; Ovalbumin, 9006-59-1; Proteins