A spectrophotometric enzyme assay watches a coloured or UV-absorbing product accumulate over time. This calculator turns that absorbance slope into proper international units, then into volume activity and specific activity, using the Beer-Lambert law and the definition of an enzyme unit.
The core calculation
By Beer-Lambert, A = ε × c × l, so a measured absorbance change corresponds
to a concentration change of ΔA / (ε × l). Dividing by the assay time gives
the rate of concentration change per minute.
Multiplying that rate by the reaction volume converts it to micromoles of product per minute, which is exactly the international unit. Dividing by the enzyme volume added gives U/mL, applying any dilution factor recovers the stock activity, and dividing by protein concentration gives specific activity in U/mg.
The full chain is:
rate (µmol/mL/min) = ΔA / (ε × l × t)
total µmol/min = rate × reaction volume (mL)
volume activity = total µmol/min / enzyme volume (mL) [U/mL]
stock activity = volume activity × dilution factor
specific activity = volume activity / protein (mg/mL) [U/mg]Worked example: a dehydrogenase assay
An assay measures NADH production at 340 nm using an extinction coefficient of 6,220 L mol⁻¹ cm⁻¹. The cuvette has a 1 cm path length and the total reaction volume is 1.0 mL. You observe a change of 0.30 absorbance units over one minute in the linear phase.
concentration change = 0.30 / (6220 × 1) = 4.82 × 10⁻⁵ mol/L/min
µmol/min in cuvette = 4.82 × 10⁻⁵ × 1000 µmol/mol × 1.0 mL / 1000
≈ 0.0482 µmol/min = 0.0482 IUIf 0.05 mL of enzyme was added to the cuvette, volume activity is:
0.0482 / 0.05 = 0.96 U/mL
If that enzyme had been diluted 10-fold before adding, the stock activity is
0.96 × 10 = 9.6 U/mL. If the protein concentration is 2.0 mg/mL in the stock,
specific activity is 9.6 / 2.0 = 4.8 U/mg.
Why specific activity matters during purification
Specific activity (U/mg) is the primary metric for tracking enzyme purification. As you move from crude extract through precipitation, column chromatography, and final polishing steps, the total units in the fraction will decrease (due to yield losses), but the specific activity should rise with each step as contaminating proteins are removed.
A purification table typically shows:
| Step | Total units | Total protein (mg) | Specific activity (U/mg) | Fold purification | Yield (%) |
|---|---|---|---|---|---|
| Crude extract | — | — | — | 1× | 100% |
| (NH₄)₂SO₄ cut | — | — | — | — | — |
| Ion exchange | — | — | — | — | — |
| Gel filtration | — | — | — | — | — |
Enter each fraction into the calculator separately to build this table row by row.
Common mistakes and how to avoid them
Using the wrong extinction coefficient: the coefficient must be for the species you are measuring at the wavelength you are using. For NADH at 340 nm, ε = 6,220 L mol⁻¹ cm⁻¹. For NADPH, the value is similar but always verify your exact wavelength and buffer conditions — pH and temperature affect measured absorbance.
Reading outside the linear phase: substrate depletion and product inhibition cause the absorbance curve to flatten. Only use the linear portion of the progress curve for the slope. In practice, keep ΔA/min below about 0.1 per minute to stay in the linear range.
Forgetting the blank: non-enzymatic reactions between substrate and coenzyme can produce a drifting background. Always run a no-enzyme blank under identical conditions and subtract its rate from the enzyme rate.