Enzyme Activity Calculator
Calculate enzyme activity (U/mL) and specific activity (U/mg) from your spectrophotometric assay data, using the Beer-Lambert law and your assay’s reaction volumes.
Calculate Enzyme Activity
Enter the absorbance change rate from your assay along with the extinction coefficient and reaction volumes to calculate enzyme activity in units per mL, and optionally specific activity in units per mg of protein.
Assay Measurements
Enter the values from your spectrophotometric assay
Activity Result
Enzyme units and specific activity
Common Assay Extinction Coefficients
Typical molar extinction coefficients used in common enzyme assays. Always confirm the exact value for your specific protocol and wavelength.
| Chromophore | Wavelength | ε (M⁻¹cm⁻¹) |
|---|---|---|
| NADH / NADPH | 340 nm | 6,220 |
| TNB (from DTNB) | 412 nm | 14,150 |
| p-Nitrophenol (pNP) | 405 nm | ~18,500 |
| DCPIP (reduced) | 600 nm | ~21,000 |
| ABTS radical cation | 414 nm | ~31,100 |
Enzyme Activity FAQ
Everything you need to know about calculating enzyme activity and specific activity.
One unit (U) of enzyme activity is defined as the amount of enzyme that catalyzes the conversion of 1 micromole of substrate per minute under specified assay conditions.
Enzyme activity (U/mL) measures total catalytic activity per volume of sample, while specific activity (U/mg) normalizes that activity to the amount of total protein present, making it useful for comparing enzyme purity between preparations.
Activity is calculated using the Beer-Lambert law: the rate of absorbance change per minute is divided by the product of the molar extinction coefficient and path length to get the reaction rate, which is then scaled by the reaction and sample volumes to get units per mL.
NADH and NADPH have a molar extinction coefficient of approximately 6220 M⁻¹cm⁻¹ at 340nm, which is commonly used in dehydrogenase assays that monitor NADH production or consumption.
If your enzyme sample was diluted before being added to the reaction, the measured activity only reflects the diluted sample. Multiplying by the dilution factor scales the result back up to represent the activity of your original, undiluted enzyme preparation.