PDH links glycolysis to the Krebs cycle, and its activity changes within minutes as the complex is switched off by phosphorylation. Here is how activity kits measure it, how to prepare samples so the number means something, and how to turn a slope into units.
Range: Biochemical and cell-based assays6 minute readUpdated
The pyruvate dehydrogenase complex sits in the mitochondrial matrix and decides whether pyruvate from glycolysis is burned in the Krebs cycle. It is a large assembly of three enzymes, E1, E2 and E3, that together carry out one reaction:
Pyruvate + CoA + NAD⁺ → acetyl-CoA + CO₂ + NADH
The reaction needs five cofactors: thiamine pyrophosphate on E1, lipoic acid on E2, FAD on E3, plus CoA and NAD⁺. Assay buffers supply the soluble ones, so the rate depends on how much active complex the sample holds.
PDH is regulated on the spot. Pyruvate dehydrogenase kinases (PDK1–4) phosphorylate the E1 subunit and switch it off; phosphatases (PDP1 and 2) switch it back on. Acetyl-CoA and NADH, its own products, also slow it. That is why PDH activity is studied in diabetes, cancer metabolism, exercise, ischaemia and inherited PDH deficiency, and why sample handling decides the result.
| Method | What is measured | Best for |
|---|---|---|
| Direct NADH | Increase in absorbance at 340 nm, or NADH fluorescence | Isolated mitochondria and purified complex |
| Dye-coupled | NADH reduces a tetrazolium through an electron carrier; formazan read in the visible range | Crude tissue homogenates and cell lysates |
| Immunocapture | Antibody-coated wells pull out PDH, then a dye-coupled read | Lysates with many competing dehydrogenases |
| Radiometric | ¹⁴CO₂ released from [1-¹⁴C]pyruvate | Reference method in specialist labs |
Reading NADH at 340 nm is simple but unreliable in crude extracts: turbidity, other NADH-producing and NADH-consuming enzymes and ultraviolet-absorbing compounds all move the baseline. Coupling NADH to a tetrazolium dye through an intermediate electron carrier moves the read into the visible range and was developed for exactly this case; the rate depended completely on pyruvate, CoA and NAD⁺, and matched other methods in both purified mitochondria and brain homogenate. Most colorimetric PDH kits use a version of this coupling.
The outline shows the logic. Use the volumes, times and wavelength in your kit manual.
The blank matters more for PDH than for most enzymes. Lysates contain other dehydrogenases and reducing compounds that reduce the dye on their own; leaving out pyruvate removes only the PDH reaction, so the difference between the wells is PDH.
For a direct NADH read in a 1 cm cuvette:
Activity (µmol/min/mL) = (ΔA340/min × Vtotal) ÷ (6.22 × Vsample)
6.22 is the millimolar extinction coefficient of NADH at 340 nm. One unit (U) is one µmol of NADH formed per minute. A worked example:
In a microplate, the path length is shorter than 1 cm and depends on the volume, and dye-coupled kits read a formazan with its own coefficient. Use the kit’s NADH or formazan standard curve to convert the slope into µmol, rather than the cuvette formula.
Per mg of protein is the usual unit. For tissues whose mitochondrial content differs, such as trained and untrained muscle, also express PDH relative to citrate synthase activity measured in the same extract, the standard marker of mitochondrial content.
Several kits have “pyruvate” in the name and measure different enzymes. Check which reaction you need before ordering:
| Enzyme | Reaction | Where | Typical readout |
|---|---|---|---|
| Pyruvate dehydrogenase (PDH) | Pyruvate → acetyl-CoA + NADH | Mitochondrial matrix | NADH increase at 340 nm, or dye-coupled |
| Pyruvate kinase (PK) | Phosphoenolpyruvate + ADP → pyruvate + ATP | Cytosol, last step of glycolysis | Coupled to LDH, NADH decrease at 340 nm |
| Pyruvate carboxylase (PC) | Pyruvate + HCO₃⁻ + ATP → oxaloacetate | Mitochondrial matrix; biotin-dependent | Coupled to malate dehydrogenase or citrate synthase |
| Lactate dehydrogenase (LDH) | Pyruvate + NADH ⇌ lactate + NAD⁺ | Cytosol | NADH change at 340 nm |
PDH feeds the Krebs cycle; succinate dehydrogenase sits inside it and in the respiratory chain, so the two are often measured together. See the SDH activity assay guide, the overview of enzyme activity assay kits for hexokinase, pyruvate kinase and others, and the lysis buffer guide for keeping enzymes active during extraction. PDH and other enzyme activity kits are in the biochemical and cell-based assay range.
The rate at which the pyruvate dehydrogenase complex in a sample converts pyruvate, CoA and NAD⁺ into acetyl-CoA and NADH. Kits read the NADH directly at 340 nm or couple it to a coloured dye, and report activity as units per mg of protein.
The usual causes are delay before freezing, which lets kinases switch PDH off, no phosphatase inhibitors in the extraction buffer, mitochondria that were not opened, or missing cofactors. Freeze tissue at once, follow the kit’s extraction method and check the positive control.
Pyruvate kinase makes pyruvate in the cytosol at the last step of glycolysis. Pyruvate dehydrogenase uses pyruvate in the mitochondria to make acetyl-CoA for the Krebs cycle. They are measured with different kits and reactions.
Yes. Harvest cells quickly, wash in cold PBS, and lyse in the kit’s buffer with phosphatase inhibitors, or snap-freeze the pellet. A few million cells usually give enough activity for a plate assay; check the kit’s sensitivity.
For research use only. Protocols here are general outlines: follow your kit manual and your institution’s safety rules. See our editorial policy.
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