This article describes a method for estimating cellular levels of pyruvate in Caenorhabditis elegans using enzyme-based colorimetric assays. The process involves preparing a cellular fraction, treating it to remove proteins, and measuring absorbance to determine pyruvate concentration.
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.
Use a colorimetric assay kit to measure the concentration of pyruvate in the test samples, and carry out duplex examinations.
Add 10 microliters of the test samples to different wells of a 96-well plate, and add 90 microliters of working reagent to each sample. Incubate covered for 30 minutes at room temperature. Then, place the plate in a microplate reader to measure the absorbance of each well at 570 nanometers.
Plot the pyruvate standard curve and calculate the pyruvate concentrations from the standard curve by multiplying each concentration by 2.25.
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