Enzyme Activity Calculator
Enter the absorbance slope ΔA/min, molar extinction coefficient ε, path length, reaction and sample volumes, and protein concentration to compute enzyme activity (U/mL) and specific activity (U/mg).
Input Data
Results
At a glance:Enzyme activity quantifies the rate of an enzyme-catalyzed reaction. One international unit (1 U) converts 1 µmol of substrate (or produces 1 µmol of product) per minute under standard conditions. By spectrophotometry, the Beer–Lambert law (A = ε·l·c) converts the absorbance change rate into product formation rate: activity (U/mL) = (ΔA/min × V_total × 10⁶) ÷ (ε × l × V_sample × 1000). Specific activity = activity ÷ protein concentration (U/mg), a key indicator of purity.
Formula
Enzyme activity: U/mL = (ΔA/min × V_total × 10⁶) ÷ (ε × l × V_sample × 1000).
10⁶ converts mol to µmol; 1000 converts mL to L (matching ε in mol/L).
Specific activity: U/mg = activity (U/mL) ÷ protein concentration (mg/mL).
$$\text{U/mL} = \dfrac{\Delta A/min \times V_{total} \times 10^{6}}{\varepsilon \times l \times V_{sample} \times 1000}$$$$\text{Specific activity} = \dfrac{\text{U/mL}}{\text{mg/mL}}$$How to Use
- Enter the slope of absorbance vs. time in the linear phase as ΔA/min, and the product molar extinction coefficient ε.
- Enter the cuvette path length (standard 1 cm), total reaction volume, and enzyme sample volume.
- Enter the protein concentration to get specific activity; the right panel shows U/mL and U/mg instantly.
Common product/substrate molar extinction coefficients (ε)
| Product / substrate | Wavelength (nm) | ε (M⁻¹cm⁻¹) |
|---|---|---|
| NADH / NADPH | 340 | 6,220 |
| p-nitrophenol (pNP) | 405 | 18,000 |
| 2-nitroaniline | 410 | 8,800 |
| Reduced cytochrome c | 550 | 21,000 |
ε varies slightly with wavelength, buffer, and temperature; cite the measured or literature value for your exact conditions.
Case Studies
Monitoring dehydrogenase via NADH
Reaction monitored at 340 nm for NADH consumption: ΔA/min = 0.05, ε = 6220, path length 1 cm, total volume 1 mL, sample 0.01 mL.
Activity = (0.05 × 1 × 10⁶) ÷ (6220 × 1 × 0.01 × 1000) ≈ 0.8039 U/mL.
Means each mL of enzyme sample catalyzes about 0.80 µmol NADH conversion per minute.
Purity assessed by specific activity
Same sample with protein concentration 2 mg/mL: specific activity = 0.8039 ÷ 2 ≈ 0.4019 U/mg.
After purification, if total activity is kept while protein drops, specific activity rises — the target enzyme is concentrated and contaminants removed.
Specific activity is the basis for purification fold and recovery; it reflects purity better than U/mL alone.
FAQ
What is one enzyme unit (U)?
The international unit 1 U is defined as the amount of enzyme that converts 1 µmol of substrate (or produces 1 µmol of product) per minute under standard conditions (specified temperature, pH, saturated substrate). The SI unit katal (kat) is 1 mol per second, so 1 U ≈ 16.67 nkat. Labs most often use U and U/mL.
Why take the slope of the linear phase?
Enzyme activity should be expressed as the initial rate, when substrate is abundant and product inhibition / reverse reaction are negligible, so absorbance vs. time is a straight line. Using the plateau (substrate depleted) underestimates true activity. Always compute ΔA/min from the still-linear segment after reaction start.
Where do I get the molar extinction coefficient ε?
ε is an intrinsic property of the product or substrate at a given wavelength; common values are in the literature (e.g. NADH at 340 nm is 6220 M⁻¹cm⁻¹). With non-standard buffers or temperatures, build your own standard curve from a known-concentration standard to obtain ε for better accuracy.
What is the difference between specific and total activity?
Activity (U/mL) is the catalytic capacity per unit volume of sample; specific activity (U/mg) is per mg of protein. Specific activity removes the effect of total protein and reflects purity — during purification, rising specific activity means contaminants are removed and the target enzyme is enriched.
What if the path length is not 1 cm?
Just enter the actual path length (cm); the formula already includes l. Microplate path lengths are often below 1 cm and vary with volume, so use the instrument's path-length correction or calibrate with a standard, or activity will be overestimated.
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References
Content review: Calculatorism Science Team. Results are for reference only; please refer to the relevant authorities for the official figures.