Enzyme Activity Calculator

Rate of absorbance change in the assay cuvette.
mM⁻¹cm⁻¹
For NADH: 6.22 mM⁻¹cm⁻¹.
mL
mL
mg/mL
Protein concentration of the enzyme sample.
Chemistry

Calculate enzyme activity (units/mL) and specific activity (units/mg protein) from an assay.

Formula

Activity = (ΔAbs/min × V_total) / (ε × V_enzyme); Specific = Activity / [protein]
  • 1 unit of enzyme activity = 1 µmol of substrate converted per minute.
  • ε for NADH = 6.22 mM⁻¹cm⁻¹ at 340 nm (most common assay dye).
  • Optical path length assumed to be 1 cm.
  • Dilution factors must be accounted for before entering concentrations.

ΔAbs = 0.1/min, ε = 6.22, 1.0 mL total, 0.1 mL enzyme, 1 mg/mL protein

Inputs
  • ΔAbsorbance per Minute: 0.1
  • Extinction Coefficient (ε): 6.22 mM⁻¹cm⁻¹
  • Total Assay Volume: 1 mL
  • Enzyme Volume in Assay: 0.1 mL
  • Protein Concentration: 1 mg/mL

Activity = (0.1 × 1.0) / (6.22 × 0.1) = 0.161 units/mL. Specific = 0.161/1 = 0.161 units/mg.

Frequently asked questions

What is a unit of enzyme activity?
One unit (U) is the amount of enzyme that catalyzes conversion of 1 µmol of substrate per minute under defined conditions.
What is specific activity?
Specific activity = units per mg of protein. It indicates enzyme purity — higher = purer preparation.