A spectrophotometric method for quantitation of carboxyl group modification of proteins using Woodward's Reagent K.
Reaction of proteins with Woodward's Reagent K in 0.05 ionic strength Tris-HCl, pH 7.8, followed by removal of excess reagent by chromatography on Sephadex G-25 in the same buffer, results in covalently attached chromophores with an absorption maximum at 340 nm and an extinction coefficient of 7000 M-1 cm-1. This absorbance can be used to quantitate the reaction of Woodward's Reagent K with carboxyl groups in proteins, provided sulfhydryl groups do not react. The chromophore also enables specific detection and identification of carboxyl-modified peptides upon separation by chromatography or electrophoresis.
D007202:Indicators and Reagents; D007555:Isoxazoles; D009113:Muramidase / Q000032:analysis; D010047:Ovalbumin / Q000032:analysis; D010751:Phosphopyruvate Hydratase / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D012441:Saccharomyces cerevisiae; D012710:Serum Albumin, Bovine / Q000032:analysis; D013053:Spectrophotometry / Q000379:methods
D007202:Indicators and Reagents; D007555:Isoxazoles; D011506:Proteins; D012710:Serum Albumin, Bovine; C009686:N-ethyl-5-phenylisoxazolium-3'-sulfonate; D010047:Ovalbumin; D009113:Muramidase; D010751:Phosphopyruvate Hydratase