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
article
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
Chemicals/CAS: ovalbumin, 77466-29-6; Carboxylic Acids; Esters; Isoxazoles; N-ethyl-5-phenylisoxazolium-3'-sulfonate, 4156-16-5; Ovalbumin, 9006-59-1; Proteins
Topics
Food technologyChemical modificationChromatographic analysisDegradationEsterspH effectsSpectrophotometryMolar extinctionProteinsChemical analysisDegradationIon exchange chromatographyMethylationMolecular stabilityPHProtein modificationProtein stabilityReaction analysisSpectrophotometryTemperature dependenceValidation processAnimalsCarboxylic AcidsChickensEstersIsoxazolesKineticsOvalbuminProteinsSpectrophotometry, Ultraviolet
TNO Identifier
237087
ISSN
0003-2700
Source
Analytical Chemistry, 75(10), pp. 2512-2516.
Pages
2512-2516
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