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

A number of relevant properties of Woodwardʹs reagent K have been determined, such as the stability of the reactant and the optimal reaction conditions of the reactant with protein carboxylates. A Woodwardʹs reagent K stock solution was stable at 4 (degree)C for prolonged time, whereas upon storage at 22 (degree)C, almost 20% of the reactive compound was lost within 1 week. The pH-dependency of the spontaneous degradation reaction of Woodwardʹs 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 Woodwardʹs 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 Woodwardʹs 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.