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 degrees C for prolonged time, whereas upon storage at 22 degrees 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.
Kosters
Hans A
HA
Wageningen Centre for Food Sciences, Diedenweg 20, 6700 AN Wageningen, The Netherlands.
de Jongh
Harmen H J
HH
D000818:Animals; D002264:Carboxylic Acids / Q000032:analysis* / Q000737:chemistry*; D002645:Chickens; D004952:Esters / Q000737:chemistry*; D007555:Isoxazoles / Q000737:chemistry*; D007700:Kinetics; D010047:Ovalbumin / Q000737:chemistry; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet
D002264:Carboxylic Acids; D004952:Esters; D007555:Isoxazoles; D011506:Proteins; C009686:N-ethyl-5-phenylisoxazolium-3'-sulfonate; D010047:Ovalbumin