Most enzyme activity kits turn a reaction you cannot see into a colour change you can measure. Once you know the two or three ways they do it, choosing a kit and reading the result gets much easier.
Range: Biochemical and cell-based assays4 minute readUpdated
An ELISA tells you how much of an enzyme is present. An activity assay, often sold as an enzymatic assay kit, tells you how much work it is doing: substrate converted per minute under defined conditions. The two often disagree, because enzymes are switched on and off by phosphorylation, cofactors, inhibitors and damage. Measure both when the question is about function, pairing activity kits with ELISA kits for the same enzyme.
Many dehydrogenases produce or consume NADH or NADPH, which absorb UV light at 340 nm while their oxidised forms do not. The absorbance change per minute gives the rate directly. You need a reader that measures at 340 nm and UV-transparent plates or quartz cuvettes.
The electrons from the reaction reduce a dye, often through an electron carrier. DCPIP loses its blue colour; tetrazolium salts such as WST, INT or MTT become coloured formazans. These readouts use visible light and standard plates.
The enzyme acts on a synthetic substrate that releases a coloured product. Many glycosidases are measured with p-nitrophenyl substrates, which release yellow p-nitrophenol, read at 400–405 nm once the reaction is stopped with alkali.
| Enzyme | Reaction measured | Typical readout |
|---|---|---|
| Succinate dehydrogenase (SDH) | Succinate → fumarate; electrons reduce DCPIP | Decrease at 600 nm |
| Pyruvate dehydrogenase (PDH) | Pyruvate + CoA + NAD⁺ → acetyl-CoA + NADH | NADH at 340 nm, or a coupled dye at about 450 nm |
| Hexokinase | Glucose → glucose-6-phosphate, coupled to G6PDH making NADPH | Increase at 340 nm |
| Pyruvate kinase | PEP → pyruvate, coupled to LDH consuming NADH | Decrease at 340 nm |
| Alpha-galactosidase (α-gal) | p-nitrophenyl-α-D-galactoside → p-nitrophenol | Increase at 405 nm after alkaline stop |
| Alanine aminotransferase (ALT, also SGPT: serum glutamic-pyruvic transaminase) | Alanine + α-ketoglutarate → pyruvate, coupled to LDH | Decrease at 340 nm |
| Thioredoxin reductase | NADPH reduces DTNB to yellow TNB | Increase at 412 nm, with an inhibitor control |
| Laccase | Oxidation of ABTS to a green radical | Increase at 420 nm |
Radical scavenging assays measure how well a sample neutralises a stable coloured radical. They report total antioxidant capacity rather than the activity of one enzyme.
Results are usually expressed as percent inhibition or as equivalents of a reference antioxidant such as Trolox.
For a complete worked example, see the SDH activity assay guide. For cell-level readouts, see the cell viability guide, and for kits, the biochemical assay range.
A ready-made set of buffer, substrate, cofactors and detection reagents that measures how fast an enzyme in your sample converts its substrate, usually by a change in absorbance per minute.
Because many enzymes are dehydrogenases or can be coupled to one, and the cofactors NADH and NADPH absorb strongly at 340 nm while their oxidised forms do not. The absorbance change follows the reaction directly.
An assay where the product of your enzyme's reaction is used by a second, helper enzyme in excess, whose reaction is easy to measure. Hexokinase, for example, is measured by coupling its product to glucose-6-phosphate dehydrogenase, which makes NADPH.
Kinetic measurement, taking several readings over time, gives the rate directly and is less affected by sample colour and turbidity. Endpoint reads are simpler and suit kits whose reaction is stopped, such as p-nitrophenol assays.
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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