Enzyme Activity Calculator

Enzyme activity is the rate at which an enzyme converts substrate into product, most commonly measured spectrophotometrically by tracking how quickly absorbance changes as a colored substrate or product is consumed or formed. Activity is expressed in units (U), where 1 U equals the amount of enzyme that converts 1 micromole of substrate per minute under the assay conditions.

Activity (U/mL) = (delta A / delta t times Vt times 1000 times DF) / (epsilon times d times Vs), where delta A / delta t is the absorbance change per minute, Vt is total reaction volume, DF is the dilution factor, epsilon is the molar extinction coefficient, d is the cuvette path length, and Vs is the sample volume added to the reaction.

Specific activity is enzyme activity divided by total protein concentration, expressed in units per milligram of protein (U/mg). It measures how concentrated the active enzyme is relative to all the protein present in a sample, making it the standard way biochemists track how much an enzyme preparation has been purified across purification steps.

One Unit (U) is formally defined as the amount of enzyme that catalyzes the conversion of 1 micromole of substrate into product per minute, under specified assay conditions such as temperature and pH. It is the standard, instrument-independent way to compare enzyme activity across labs and experiments.

The Michaelis-Menten Equation Calculator models reaction velocity as a function of substrate concentration, v = Vmax[S] / (Km + [S]), and requires you to already know Vmax and Km. This Enzyme Activity Calculator instead computes activity directly from a real absorbance-based assay measurement, the rate of change in absorbance over time, converting that raw instrument reading into U/mL and U/mg. They answer different questions: one describes reaction kinetics theoretically, the other measures how much active enzyme is actually present in a sample.

Use the molar extinction coefficient of the specific chromophore your assay produces or consumes at the wavelength you are measuring. A very common example is NADH at 340 nm, with an extinction coefficient of about 6220 M^-1 cm^-1, used in many dehydrogenase assays such as LDH and MDH.

Total reaction volume (Vt) is the full assay volume in the cuvette, used to convert the measured rate into total micromoles of product formed per minute. Sample volume (Vs) is only the portion of that total volume contributed by your enzyme sample, used to express the final activity per milliliter of the original enzyme sample rather than per milliliter of the whole diluted reaction mixture.

The dilution factor accounts for any dilution of the original enzyme sample before it was added to the assay. For example, if you diluted your enzyme stock 1 in 10 before pipetting it into the reaction, enter a dilution factor of 10 so the calculated activity reflects the concentration in the original, undiluted stock.

No, activity as reported by this calculator is always a positive number representing a rate of catalysis. Some assays measure a decrease in absorbance (for example NADH consumption), but you should enter the magnitude of that change, not a negative number, since the calculator reports the rate of the reaction, not its direction.

Divide the activity in U/mL by the total protein concentration of the sample in mg/mL, giving specific activity in U/mg. This calculator performs that division automatically once you enter the protein concentration, typically measured separately using a Bradford, BCA, or Lowry protein assay.

Specific activity depends heavily on assay conditions (temperature, pH, substrate concentration, buffer composition) and on how pure your enzyme preparation is, so direct comparison to a different lab's published value is only meaningful if assay conditions closely match. A low specific activity relative to a purified reference usually indicates a crude or impure sample; specific activity should increase at each purification step for the same enzyme.