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 degreesC for prolonged time, whereas upon storage at 22 degreesC, 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.