Enzyme Activity Calculator — Spectrophotometric Assay (U/mL)
Determine how much enzyme activity (in international units per mL) is present in your sample from a continuous spectrophotometric assay. Enter the absorbance change rate (ΔA/min), the assay and sample volumes, the molar extinction coefficient of the product (or substrate), and the cuvette path length.
mL
mL
M⁻¹cm⁻¹
cm
mg/mL
1 U = 1 µmol of substrate converted per minute
- 1
ΔA × V_total × 1000 (numerator)
0.1 × 1 × 1000 = 100 - 2
ε × l × V_sample (denominator)
6,220 × 1 × 0.1 = 622 - 3
Enzyme activity
100 ÷ 622 = 0.16081 U = 1 µmol of substrate converted per minute.
How does this calculator work?
Enzyme activity (U/mL) = (ΔA/min × V_total × 1000) ÷ (ε × l × V_sample). Specific activity (U/mg) divides that by the protein concentration. 1 U = 1 µmol substrate converted per minute. Requires a linear reaction phase and the Beer-Lambert ε for your measured compound.
Formula
How this is calculated
A continuous spectrophotometric enzyme assay monitors absorbance at a fixed wavelength as the enzyme converts substrate to product (or consumes a cofactor such as NADH). The Beer-Lambert law relates absorbance to concentration: A = ε × c × l, where ε is the molar extinction coefficient (M⁻¹ cm⁻¹), c is concentration (M), and l is path length (cm). Rearranging gives the rate of change of concentration: Δc/min = ΔA/min ÷ (ε × l) in mol/L/min.
To get activity in the original enzyme sample, scale by the total assay volume and divide by the sample volume: Activity (U/mL) = ΔA/min × V_total × 1000 / (ε × l × V_sample). The factor 1000 converts mol to µmol (1 U = 1 µmol/min) and millilitres to litres simultaneously. Common extinction coefficients (at their standard wavelength): NADH/NADPH at 340 nm = 6,220 M⁻¹cm⁻¹; p-nitrophenol at 405 nm = 18,500; DTNB at 412 nm = 14,150.
Specific activity (U/mg) divides the volumetric activity by the protein concentration of the enzyme preparation, and is used to compare enzyme purity across purification steps. The calculator assumes a linear reaction rate (the assay is in the linear phase) and a constant path length — both of which should be verified experimentally. Non-linear traces (burst kinetics, product inhibition, substrate depletion) require more advanced analysis.
Frequently asked questions
1 enzyme unit (U) is the amount of enzyme that converts 1 µmol of substrate per minute under defined conditions (pH, temperature, substrate concentration). The SI unit katal (kat) = 1 mol/s is less commonly used in biochemistry; 1 U = 16.67 nkat.
Use the published ε for the compound you are monitoring at your specific wavelength. The most common reference values (at 25°C, aqueous buffer): NADH at 340 nm = 6,220 M⁻¹cm⁻¹; NADPH at 340 nm = 6,220; p-nitrophenol at 405 nm = 18,500; DTNB (Ellman reagent) at 412 nm = 14,150.
The Beer-Lambert formula assumes a constant rate (ΔA/min). If the substrate is depleted, product inhibition occurs, or enzyme is inactivated, the rate is no longer linear and the calculated activity will be underestimated. Always verify linearity by examining the full absorbance-vs-time trace before reading ΔA/min.
Also known as
TG we-Calculate Editorial Team. (2026). Enzyme Activity Calculator — Spectrophotometric Assay (U/mL) [Online calculator]. TG we-Calculate. https://we-calculate.com/calculator/enzyme-activity-calculator
TG we-Calculate Editorial Team. "Enzyme Activity Calculator — Spectrophotometric Assay (U/mL)." TG we-Calculate. 2026. https://we-calculate.com/calculator/enzyme-activity-calculator.
TG we-Calculate Editorial Team, "Enzyme Activity Calculator — Spectrophotometric Assay (U/mL)," TG we-Calculate, 2026. [Online]. Available: https://we-calculate.com/calculator/enzyme-activity-calculator
@misc{wecalculate_enzyme_activity_calculator, title = {Enzyme Activity Calculator — Spectrophotometric Assay (U/mL)}, author = {{TG we-Calculate Editorial Team}}, howpublished = {\url{https://we-calculate.com/calculator/enzyme-activity-calculator}}, year = {2026}, note = {TG we-Calculate} }
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