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Enzyme Activity Calculator (Units, Specific Activity)

Calculate enzyme activity in U/mL from the rate of absorbance change, specific activity per mg of protein, a reaction rate, and conversions between units and katal.

Formula

activity (U/mL)=(ΔA/min)×Vassayε ℓ Venzyme×DF\text{activity}\,(\mathrm{U/mL}) = \dfrac{(\Delta A/\mathrm{min})\times V_{\text{assay}}}{\varepsilon\,\ell\,V_{\text{enzyme}}}\times\mathrm{DF}
specific activity (U/mg)=activity (U/mL)protein (mg/mL)\text{specific activity}\,(\mathrm{U/mg}) = \dfrac{\text{activity}\,(\mathrm{U/mL})}{\text{protein}\,(\mathrm{mg/mL})}
1 U=1 μmol/min=16.67 nkat1\ \mathrm{U} = 1\ \mu\mathrm{mol/min} = 16.67\ \mathrm{nkat}
ΔA/min\Delta A/\mathrm{min}
initial rate of change of absorbance, corrected for the blank
ε\varepsilon
extinction coefficient of the chromophore, in mM⁻¹ cm⁻¹
Vassay, VenzymeV_{\text{assay}},\ V_{\text{enzyme}}
total reaction volume and the volume of enzyme solution added

How it works

An enzyme unit (U) is the amount that converts one micromole of substrate per minute under stated conditions. If the reaction produces or consumes something that absorbs light, the rate of change of absorbance, through the Beer-Lambert law, gives the rate of change of concentration, and from it the micromoles converted per minute in the cuvette. Dividing by the volume of enzyme solution that was added gives the activity per millilitre.

Specific activity divides that by the protein concentration, and is the usual measure of the purity of an enzyme preparation: it rises as the enzyme is purified. The katal is the SI unit, one mole per second, so 1 U is 16.67 nanokatal.

Worked example

NADH consumption at 340 nm (ε = 6.22 mM⁻¹ cm⁻¹): ΔA = 0.12 per minute in a 3.0 mL assay in a 1 cm cuvette, with 0.05 mL of enzyme.

  1. Rate in the cuvette = 0.12 / 6.22 = 0.0193 mM/min, times 3.0 mL = 0.0579 µmol/min.
  2. Per mL of enzyme solution: 0.0579 / 0.05 = 1.158 U/mL.

1.158 U/mL, or 19.3 nkat/mL.

These are the values the calculator opens with, so you can check its output against this example.

Assumptions

  • The rate is the initial, linear rate, with substrate not depleting.
  • The blank (non-enzymatic) rate has been subtracted.
  • The extinction coefficient applies at the pH, temperature and wavelength used.

Common mistakes

  • Using a rate measured on a curving progress curve instead of the initial slope.
  • Comparing units from assays run at different temperatures or pH.
  • Forgetting the dilution factor of the enzyme, or mixing millilitres and microlitres.

Related tools

Michaelis-Menten CalculatorRate at a given [S], or [S] for a target rate, with the curveOpen calculatorEnzyme Kinetics Calculator (Km, Vmax Fit)Nonlinear Michaelis-Menten fit, with Lineweaver-Burk for comparisonOpen calculatorBeer-Lambert Law CalculatorAbsorbance, concentration or ε, and absorbance ⇄ % transmittanceOpen calculatorProtein Concentration Calculator (A280, A205)Concentration from absorbance, and mg/mL ⇄ µMOpen calculator

Related equipment

Service documentation, failure modes and parts for the instruments this calculation is used with.