Enzyme Activity Calculator
Enzyme activity from the rate of absorbance change in a spectrophotometric assay — in units per millilitre of enzyme and, with the protein concentration, units per milligram.
One unit (U) converts one micromole of substrate per minute.
How the enzyme activity calculator works
One unit (U) converts one micromole of substrate per minute. Beer–Lambert turns the absorbance change per minute into a concentration change: ΔA ÷ (ε × l) mol/L/min. Multiply by the assay volume for micromoles per minute in the cuvette, then divide by the volume of enzyme you added (and multiply by any dilution) for U/mL. Divide by the protein concentration for specific activity, the measure of purity.
Formula: U/mL = ΔA/min × V_assay × dilution × 10⁶ ÷ (ε × l × V_enzyme); specific activity = U/mL ÷ mg/mL
Worked examples
| Inputs | Activity | Note |
|---|---|---|
| ΔA 0.12/min with NADH, 10 µL of enzyme in 1 mL | 1.929 U/mL | 1.93 U/mL, 0.96 U/mg |
| p-Nitrophenol assay, 20 µL of a 1:10 dilution | 8.333 U/mL | 8.33 U/mL |
| Microplate: 0.2 mL, 0.5 cm path | 0.643 U/mL | 0.64 U/mL |
FAQFrequently asked questions
What is a unit?
The international unit: one micromole of substrate converted per minute under the stated conditions. The SI katal (mol/s) exists but units are what suppliers and papers use; 1 U = 16.67 nkat.
Why does specific activity matter?
It is activity per milligram of protein, so it rises as an enzyme is purified and falls as it denatures. Comparing it with the supplier’s or the literature value tells you how good your preparation is.
What if the absorbance goes down?
Assays that consume NADH read a falling absorbance; the calculator uses the magnitude of the slope, so enter the change as measured.
Why keep the rate linear?
The initial rate is what reflects the enzyme; as substrate is used up or product inhibits, the slope bends. Use the first minute or two while the trace is straight, and keep ΔA under about 0.5 per minute.
Where these figures come from
- NCBI Bookshelf — Molecular Biology of the Cell — growth kinetics and molecular conventions
- Wellcome Sanger Institute — a leading UK genomics institute
Last checked: September 2026. Formulas are the standard textbook forms; assumptions are stated on each page because they are where these models break.