Substrate cycling based fluorometric assay for dihydroxyacetone phosphate

• Dihydroxyacetone phosphate (DHAP) was assayed using a new fluorescence-based enzymatic assay.
• DHAP quantitation was achieved in 96-well microplates through a multi-enzymatic based amplification system.
• DHAP was continuously recycled using α-glycerophosphate dehydrogenase and α-glycerophosphate oxidase enzymes.

A sensitive fluorometric assay for the determination of dihydroxyacetone phosphate (DHAP) is reported here. DHAP is reduced to l-glycerol-3-phosphate with NADH-dependent α-glycerophosphate dehydrogenase. DHAP recycling is provided by oxidation reaction catalysed by α-glycerophosphate oxidase to release hydrogen peroxide. The reaction of hydrogen peroxide with Amplex® Red reagent under horseradish peroxidase catalysis leads to the fluorescent product resorufin. The limit of detection of DHAP is estimated at 1 pmol which is roughly 2250 fold more sensitive than the usual DHAP assay based on the detection of NADH by spectrophotometry. This assay is ready-to-use for automated medium-throughput screening.

α-GPDH: α-glycerophosphate dehydrogenase; α-GPO: α-glycerophosphate oxidase; HRP: horseradish peroxidase.Figure optionsDownload as PowerPoint slide