Enzyme activity assays
The product, or a dye it generates, absorbs visible or UV light, and the rate is read as a change in absorbance per minute: p-nitrophenol at 405 nm from phosphatase and esterase substrates, NADH at 340 nm for dehydrogenases. Robust, cheap and linear to an absorbance of about 1.5–2, with the extinction coefficient and the well's path length converting it to µM/min.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
Colorimetric
The product, or a dye it generates, absorbs visible or UV light, and the rate is read as a change in absorbance per minute: p-nitrophenol at 405 nm from phosphatase and esterase substrates, NADH at 340 nm for dehydrogenases. Robust, cheap and linear to an absorbance of about 1.5–2, with the extinction coefficient and the well's path length converting it to µM/min.
How it differs from the standard method
- Sensitivity is set by the extinction coefficient: p-nitrophenolate at pH 10 is about 18 000 M⁻¹ cm⁻¹, NADH about 6220 M⁻¹ cm⁻¹.
- Stray light caps usable absorbance near 2, so a fast well can bend for reasons that have nothing to do with the enzyme.
- The plate path length (about 0.62 cm for 200 µL in a 96-well plate) has to be used, or measured with a path-check, when converting to concentration.
- Coloured or turbid samples, such as crude lysates, need their own sample blanks.
1A rate, not an amount
An enzyme activity assay measures how fast an enzyme turns substrate into product. The enzyme is mixed with its substrate under defined conditions (buffer, pH, temperature, cofactors) and something that changes as product appears is followed over time: the absorbance of a coloured product, the fluorescence of a released fluorophore, or the appearance of NADH or NADPH made by a second, reporting enzyme.
2Initial rate versus endpoint
A progress curve is straight only while the substrate concentration has barely changed. As substrate is consumed the enzyme slows, and the curve bends towards a plateau.
Titrate the enzyme
Find the amount that gives a clear, straight progress curve with under 10% substrate used in the window.
Blank
Substrate without enzyme at every concentration, read over the same time: spontaneous hydrolysis is real.
Pre-warm
Plate, buffer and substrate at temperature; start with enzyme so time zero is real.
Read
A read every few seconds shows the curve was straight; a stopped read has to rely on a separate time course.
Fit
Substrate from well below to well above Km, fitted to the Michaelis–Menten equation directly.
3Continuous and stopped assays
In a continuous assay the plate reader or spectrophotometer reads every well repeatedly while the reaction runs, so each well is a curve and you can see whether it was straight. In a stopped (discontinuous) assay the reaction is quenched at a fixed time (with acid, base, a chelator or heat) and read once.
4What a single concentration can and cannot tell you
The Michaelis–Menten equation, v = Vmax·[S]/(Km + [S]), has two unknowns. One rate at one substrate concentration is a single equation in two unknowns: a high Vmax with a high Km and a low Vmax with a low Km both fit it.
- Km
- The substrate concentration at which the rate is half of Vmax. Not, in general, a dissociation constant, although it approaches one for simple mechanisms.
- Vmax and kcat
- The rate at saturating substrate, and that rate divided by the concentration of active enzyme: turnovers per second per active site.
- kcat/Km
- The specificity constant: the rate constant at low substrate, and the right number for comparing substrates or enzymes.
- Coupled assay
- A reaction whose product is invisible, converted by an excess of a second enzyme into something that can be read. The reporter must never be the slow step.
- Inner-filter effect
- Loss of fluorescence because something in the well, often the substrate itself, absorbs the excitation or emission light. It makes the same product read dimmer at high substrate.
5Reading an inhibitor's mechanism
An inhibitor is characterised by measuring the full substrate series with and without it and seeing which fitted parameter moves. A competitive inhibitor binds where the substrate does, so enough substrate outcompetes it: Km rises by (1 + [I]/Ki) and Vmax is unchanged.
6Blanks and conversions
Many substrates make product without any enzyme: p-nitrophenyl phosphate hydrolyses at alkaline pH, peptide-coumarin substrates degrade slowly, and reagents for coupled assays can carry traces of the intermediate. That drift is proportional to substrate and time, so it inflates the fastest wells most and pushes Vmax up.
Key terms
Practise these as flashcards →The slope of the progress curve while substrate depletion is negligible (under about 10%). The only rate that obeys Michaelis–Menten kinetics.
Product against time for one reaction. Straight at the start, bending as substrate is used, product accumulates or the enzyme loses activity.
Initial rate per mass of protein, usually µmol/min/mg. The standard unit for following a purification.
Non-linear least-squares fit of rates against substrate to v = Vmax[S]/(Km + [S]). Preferred over linearised plots.
A well with substrate and everything except enzyme, at each substrate concentration, read through the same time course.
The reporting enzyme in a coupled assay, added in large excess so its lag is a few percent of the window and its capacity far above the fastest rate.
The dissociation constant of the inhibitor from the enzyme (or the enzyme–substrate complex). Obtained from how the apparent Km or Vmax changes with inhibitor concentration.
The depth of liquid the light passes through. 200 µL in a 96-well plate is about 0.62 cm, so a plate absorbance is not a cuvette absorbance.
When to use it
- Characterising a purified enzyme: Km, kcat and kcat/Km for its substrates, under defined conditions that someone else can reproduce.
- Following an enzyme through a purification, where specific activity tells you whether each step enriched it or lost it.
- Measuring an inhibitor's potency and mechanism, which needs full substrate series with and without inhibitor rather than one rate.
- Comparing activity between samples (cell lysates, mutants, treated and untreated) with a fixed substrate concentration once the assay has been shown to be linear.
Compared with alternatives
The instrument side of the same measurement: gain, path length, filters and kinetic reads; read it before setting up a plate-based kinetic assay.
Measures binding of a substrate analogue or inhibitor directly, with on- and off-rates, rather than its effect on turnover; prefer it when binding and catalysis need to be separated.
Gives the affinity and thermodynamics of inhibitor binding with no reporter at all, and can follow some reactions by their heat; prefer it when no optical readout exists.
Can follow an unlabelled substrate and product directly when no chromogenic or fluorogenic substrate exists, at the cost of a stopped, lower-throughput assay.
Measures how much enzyme protein is present, not how active it is; the two together give specific activity per molecule.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
The product, or a dye it generates, absorbs visible or UV light, and the rate is read as a change in absorbance per minute: p-nitrophenol at 405 nm from phosphatase and esterase substrates, NADH at 340 nm for dehydrogenases. Robust, cheap and linear to an absorbance of about 1.5–2, with the extinction coefficient and the well's path length converting it to µM/min.
- Sensitivity is set by the extinction coefficient: p-nitrophenolate at pH 10 is about 18 000 M⁻¹ cm⁻¹, NADH about 6220 M⁻¹ cm⁻¹.
- Stray light caps usable absorbance near 2, so a fast well can bend for reasons that have nothing to do with the enzyme.
- The plate path length (about 0.62 cm for 200 µL in a 96-well plate) has to be used, or measured with a path-check, when converting to concentration.
- Coloured or turbid samples, such as crude lysates, need their own sample blanks.