An enzyme-coupled assay for glyoxylic acid.
Herein, we present a new enzyme-linked spectrophotometric assay for glyoxylate that detects glyoxylate via the formation of an intensely colored formazan. This glyoxylate-specific assay is reliant upon the enzymatic conversion of glyoxylate to oxaloacetate coupled to the reduction of oxidized nicotinamide adenine dinucleotide to reduced nicotinamide adenine dinucleotide (NADH). The NADH-dependent reduction of a tetrazolium to the formazan enables the measurement of nanomole quantities of glyoxylate in an assay that is amenable to high-throughput screening methods. Assay validation was accomplished using two methods for glyoxylate generation, the base-catalyzed N-dealkylation of alpha-hydroxyhippurate to benzamide and glyoxylate and the oxidative cleavage of the glycyl Calpha-N bond in N-benzoylglycine (hippurate) by peptidylglycine alpha-amidating monooxygenase to again yield benzamide and glyoxylate. For both reactions, analysis of benzamide produced by reverse-phase high-performance liquid chromatography compared with glyoxylate measured using our glyoxylate assay showed a 1:1 molar ratio of benzamide to glyoxylate. These results indicate that the enzyme-linked spectrophotometric assay can quantitatively measure submicromole quantities of glyoxylate.
Carpenter
Sarah E
SE
Department of Chemistry, University of South Florida, Tampa, FL 33620, USA.
Merkler
David J
DJ
D002851:Chromatography, High Pressure Liquid; D006038:Glyoxylates / Q000032:analysis*; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000378:metabolism; D008292:Malate Synthase / Q000378:metabolism; D013053:Spectrophotometry / Q000379:methods*
D006038:Glyoxylates; D008291:Malate Dehydrogenase; D008292:Malate Synthase; C031150:glyoxylic acid