What this calculator does
Enzyme activity measures how fast an enzyme converts substrate, expressed in units, where one unit (U) is the amount of enzyme that converts one micromole of substrate per minute under the assay conditions. Most lab assays never measure substrate directly; they track a change in absorbance over time, using a molecule that absorbs light in proportion to how much product has formed.
The Beer-Lambert law connects absorbance to concentration through the molar absorptivity of the coloured product and the path length of the cuvette. Rearranging it turns a rate of absorbance change into a rate of concentration change, and multiplying that by the reaction volume gives the total amount of substrate converted per minute, which is the enzyme activity.
The formula
Divide the change in absorbance by the time interval to get the rate of absorbance change, then divide by the molar absorptivity and path length to convert that into a concentration change rate. Multiplying by the reaction volume and 1,000 converts mol/min into µmol/min, which is the definition of a unit of activity. If a protein mass is entered, activity is also divided by that mass to give specific activity in U/mg.
| Term | Meaning |
|---|---|
| U (unit) | The amount of enzyme that converts 1 µmol of substrate per minute. |
| ε (molar absorptivity) | How strongly the product absorbs light at the assay wavelength, in M⁻¹cm⁻¹. |
| Specific activity | Activity divided by the total protein present, in U/mg, used to compare enzyme purity between preparations. |
The inputs explained
| Field | What to enter |
|---|---|
| Change in absorbance (ΔA) | The change in absorbance measured over the time interval, read from the spectrophotometer trace. |
| Time interval (Δt) (min) | The time interval over which that absorbance change was measured, usually the linear part of the trace. |
| Total reaction volume (mL) | The total volume of the reaction mixture in the cuvette. |
| Molar absorptivity ε (M⁻¹cm⁻¹) | The molar absorptivity of the product at the assay wavelength; 6,220 M⁻¹cm⁻¹ is the commonly used value for NADH at 340 nm. |
| Path length (cm) | The path length of the cuvette, almost always 1 cm for a standard cuvette. |
| Protein in reaction (0 to skip specific activity) (mg) | The total protein in the reaction, if known, to also report specific activity. Leave at 0 to skip it. |
When to use it
Running a kinetic spectrophotometric assay
NADH-linked assays and similar coupled reactions are read as a continuous absorbance trace; the linear slope over the first minute or two is the ΔA/Δt used here.
Comparing enzyme preparations
Specific activity (U per mg of protein) is the standard way to compare how active a purified enzyme prep is against a cruder extract, independent of how much total protein each contains.
Choosing an extinction coefficient
Different chromogenic or coupled-assay products have different molar absorptivities; using the correct published value for the specific assay is essential, since activity scales directly with it.
Worked examples
Every figure in the tables below is produced by this page’s own calculator at build time, so the numbers and the tool always agree. Select any row to load that scenario.
How activity changes with the molar absorptivity used
The same absorbance trace, converted using a range of molar absorptivity values.
| Molar absorptivity ε | Enzyme activity | Activity per mL of reaction |
|---|---|---|
| 3,000 M⁻¹cm⁻¹ | 0.0500 U (µmol/min) | 0.0500 U/mL |
| 4,000 M⁻¹cm⁻¹ | 0.0375 U (µmol/min) | 0.0375 U/mL |
| 6,220 M⁻¹cm⁻¹ | 0.0241 U (µmol/min) | 0.0241 U/mL |
| 8,000 M⁻¹cm⁻¹ | 0.0188 U (µmol/min) | 0.0188 U/mL |
| 10,000 M⁻¹cm⁻¹ | 0.0150 U (µmol/min) | 0.0150 U/mL |
| 15,000 M⁻¹cm⁻¹ | 0.0100 U (µmol/min) | 0.0100 U/mL |
How activity changes with the measured absorbance change
A fixed assay setup, across a range of measured absorbance changes.
| Change in absorbance (ΔA) | Enzyme activity | Rate of absorbance change |
|---|---|---|
| 0.05 | 0.0080 U (µmol/min) | 0.0500 A/min |
| 0.10 | 0.0161 U (µmol/min) | 0.1000 A/min |
| 0.15 | 0.0241 U (µmol/min) | 0.1500 A/min |
| 0.20 | 0.0322 U (µmol/min) | 0.2000 A/min |
| 0.30 | 0.0482 U (µmol/min) | 0.3000 A/min |
Questions
What counts as one unit (U) of enzyme activity?
One unit is defined as the amount of enzyme that converts one micromole of substrate to product per minute, under the specific conditions of temperature, pH and substrate concentration used in the assay. It is not an absolute property of the enzyme; it depends on the assay conditions.
Why does the formula need the molar absorptivity of the product, not the substrate?
The assay tracks whichever species absorbs light at the chosen wavelength, and that is usually the product (or a coupled indicator dye), not the original substrate. Using the wrong molar absorptivity, or the substrate's value instead of the product's, gives an activity that is off by whatever ratio separates the two.
Why use specific activity instead of raw activity?
Raw activity depends on how much total enzyme-containing material is in the tube, which varies between preparations. Specific activity divides that out by the protein mass present, giving a figure that reflects the intrinsic activity of the enzyme itself, useful for tracking purification progress.
Does the reaction rate stay constant over the whole assay?
No. Enzyme kinetics are usually only linear early in the reaction, before substrate is depleted or product inhibition sets in. The ΔA/Δt used here should come from that initial linear portion of the trace, not the whole run.
For the Beer-Lambert relationship this calculator relies on, see the Beer-Lambert law calculator. To convert a measured rate into a rate constant instead of an activity figure, see the rate constant calculator.