Pyruvate Assay for Estimation of Pyruvate Levels in Cellular Fractions of Worms
Transcrição
Cellular levels of pyruvate — a key intermediate in energy-producing biochemical pathways — can be estimated using enzyme-based colorimetric assays.
To determine pyruvate levels in the cells of Caenorhabditis elegans — a non-parasitic nematode — take a cellular fraction prepared from adult worms containing pyruvate. Treat the solution, removing cellular proteins, and collect the clarified cellular fraction — the test sample — containing pyruvate. The absence of cellular proteins ensures an accurate determination of pyruvate levels.
Start by adding a working reagent containing the enzymes pyruvate oxidase and horseradish peroxidase, and a chromogenic dye. Incubate the mixture in the dark for an adequate duration.
During incubation, pyruvate oxidase reacts with pyruvate — its substrate — generating hydrogen peroxide as a byproduct. The absence of light prevents the spontaneous decomposition of hydrogen peroxide.
Horseradish peroxidase catalyzes the reaction between the hydrogen peroxide and the chromogenic dye, producing a colored product. The intensity of the colored reaction product is directly proportional to the pyruvate concentration in the sample.
Upon completion of the incubation period, place the sample inside a plate reader and measure the absorbance. Generate a standard curve using a serial dilution with known pyruvate concentrations.
Plot the measured absorbance value of the test sample on the curve to determine the pyruvate concentration in the test sample.
