# Enzyme Activity Calculator URL: https://speedycalculator.com/calculators/chemistry/enzyme-activity/ Last updated: 2026-10-30 > The enzyme activity calculator finds how many milligrams of an enzyme preparation you need for a working solution of a given activity – or solves for the activity, volume or specific activity instead – and converts a spectrophotometric assay rate into units per millilitre and per milligram. ## Inputs - What do you want to find? - Desired activity concentration (U/mL, U/L, U/µL, nkat/mL, µkat/mL) - Specific activity of the stock enzyme (U/mg, U/µg, U/g, nkat/mg, µkat/mg) - Final volume of the solution (mL, µL, L) - Mass of enzyme (mg, µg, g) - Absorbance change per minute (ΔA/min) - Molar absorptivity ε of the measured species (M⁻¹·cm⁻¹) — NADH or NADPH at 340 nm: 6220 - Path length (cm, mm) - Total assay volume (mL, µL, L) - Volume of enzyme sample in the assay (µL, mL) - Dilution factor of the enzyme sample - Protein concentration of the undiluted sample (0 if unknown) (mg/mL, µg/mL, g/L) ## Results - Mass of enzyme needed - Activity concentration of the solution - Final volume - Specific activity of the stock - Total enzyme activity in the solution - Rate of concentration change in the cuvette - Activity in the cuvette - Activity of the original sample - Specific activity of the sample ## Formula ``` m(enzyme) = A(desired) × V(final) / SA(stock) · total units = A × V · 1 U = 1 µmol/min = 16.67 nkat · assay: A (U/mL) = (ΔA/min) / (ε·l) × V(total) × 1000 / V(enzyme) × DF · SA = A / [protein] ``` ## Explanation Enzymes are sold and used by activity rather than by weight, because two batches of the same protein can differ widely in how much of it is active. One enzyme unit (U) is the amount of enzyme that converts one micromole of substrate per minute under defined conditions; the SI unit, the katal, is one mole per second, so 1 U = 16.67 nkat. The specific activity of a preparation – units per milligram of protein – is printed on the vial and rises as an enzyme is purified. Making up a working solution is a simple proportion: the total number of units you need is the desired activity concentration times the final volume, and dividing that by the specific activity of the stock gives the mass of powder to weigh out. The same relation can be rearranged to find the volume a given mass will make, the activity you will get, or the specific activity of the stock. To measure activity yourself, a continuous spectrophotometric assay follows the change in absorbance per minute. The Beer–Lambert law converts that slope into a concentration change (dividing by the molar absorptivity ε and the path length), and scaling by the assay volume, the volume of enzyme added and any dilution gives units per millilitre of the original sample. NADH-linked assays at 340 nm, with ε = 6 220 M⁻¹ cm⁻¹, are the classic example. ### How to use the Enzyme Activity Calculator - **What do you want to find?** - **Desired activity concentration** - **Specific activity of the stock enzyme** - **Final volume of the solution** - **Mass of enzyme** - **Absorbance change per minute (ΔA/min)** - **Molar absorptivity ε of the measured species** — NADH or NADPH at 340 nm: 6220 - **Path length** - **Total assay volume** - **Volume of enzyme sample in the assay** - **Dilution factor of the enzyme sample** - **Protein concentration of the undiluted sample (0 if unknown)** Results update as you type, every field has its own unit menu, and everything is calculated in your browser. ### How it works Pick what you want to find. For working solutions the calculator uses mass = activity × volume ÷ specific activity and its rearrangements. For an assay it divides ΔA/min by ε·l to get mol/L per minute, multiplies by the assay volume to get µmol/min, divides by the enzyme volume and multiplies by the dilution factor; dividing by the protein concentration gives the specific activity. ### Worked example **Working solution.** You need 25 mL of an enzyme solution at 5 U/mL and the stock powder has a specific activity of 40 U/mg. Total activity = 5 × 25 = 125 U (= 2 083 nkat); mass = 125 / 40 = 3.125 mg. Weigh 3.125 mg and make up to 25 mL. **Assay.** An NADH-linked assay at 340 nm gives ΔA/min = 0.12 in a 1 cm cuvette with 1 mL total volume, using 20 µL of the sample diluted 1 : 10. Rate = 0.12 / (6220 × 1) = 1.929 × 10⁻⁵ M/min = 19.29 µmol/(L·min); activity in the cuvette = 19.29 × 0.001 L = 0.01929 µmol/min = 0.01929 U. Per mL of the diluted sample: 0.01929 / 0.020 = 0.9646 U/mL; in the original sample: × 10 = 9.646 U/mL. With 2 mg/mL protein, the specific activity is 9.646 / 2 = 4.82 U/mg. ### Activity units at a glance UnitDefinitionIn UU (international unit)1 µmol/min1mU1 nmol/min0.001katal1 mol/s6 × 10⁷µkat1 µmol/s60nkat1 nmol/s0.06 ### Tips - Keep enzyme and substrate concentrations in the same unit. - Check that your data lie in the linear range of the standard curve. - pH strongly affects enzyme activity and protein solubility. - Everything is calculated in your browser. ### Common mistakes to avoid - Extrapolating a calibration curve far beyond the standards. - Mixing units (U, kat, µmol/min). - Ignoring dilution factors when reporting the original concentration. ### Inputs explained FieldWhat to enterWhat do you want to find?Choose one: Mass of enzyme to weigh out, Activity of the solution, Final volume, Specific activity of the stock …Desired activity concentrationUnits: U/mL, U/L, U/µL, nkat/mL, µkat/mLSpecific activity of the stock enzymeUnits: U/mg, U/µg, U/g, nkat/mg, µkat/mgFinal volume of the solutionUnits: mL, µL, LMass of enzymeUnits: mg, µg, gAbsorbance change per minute (ΔA/min)A numberMolar absorptivity ε of the measured speciesIn M⁻¹·cm⁻¹. NADH or NADPH at 340 nm: 6220Path lengthUnits: cm, mmTotal assay volumeUnits: mL, µL, LVolume of enzyme sample in the assayUnits: µL, mLDilution factor of the enzyme sampleA numberProtein concentration of the undiluted sample (0 if unknown)Units: mg/mL, µg/mL, g/L ### What each result means - **Mass of enzyme needed** = [Desired activity concentration]×[Final volume of the solution]÷[Specific activity of the stock enzyme] - **Activity concentration of the solution** = [Mass of enzyme]×[Specific activity of the stock enzyme]÷[Final volume of the solution] - **Final volume** = [Mass of enzyme]×[Specific activity of the stock enzyme]÷[Desired activity concentration] - **Specific activity of the stock** = [Desired activity concentration]×[Final volume of the solution]÷[Mass of enzyme] - **Rate of concentration change in the cuvette** = [Absorbance change per minute (ΔA÷min)]÷([Molar absorptivity ε of the measured species]×[Path length])×1e6 - **Activity in the cuvette** = [Absorbance change per minute (ΔA÷min)]÷([Molar absorptivity ε of the measured species]×[Path length])×[Total assay volume]×1000 - **Activity of the original sample** = [Absorbance change per minute (ΔA÷min)]÷([Molar absorptivity ε of the measured species]×[Path length])×[Total assay volume]×1000÷[Volume of enzyme sample in the assay]×[Dilution factor of the enzyme sample] ## Worked example Inputs — What do you want to find?: Mass of enzyme to weigh out; Desired activity concentration: 5 U/mL; Specific activity of the stock enzyme: 40 U/mg; Final volume of the solution: 25 mL. Results — **Mass of enzyme needed = 3.125 mg**; Total enzyme activity in the solution = 125 U (µmol/min). ## Key terms - **Enzyme unit (U)**: Enzyme amount that converts 1 µmol of substrate per minute under defined conditions. - **Katal (kat)**: SI unit of catalytic activity, 1 mol of substrate per second; 1 U = 16.67 nkat. - **Specific activity**: Units of activity per milligram of protein. - **Molar absorptivity (ε)**: Absorbance of a 1 M solution in a 1 cm cell, in M⁻¹ cm⁻¹; links absorbance to concentration via A = ε·c·l. ## FAQ **How do you calculate how much enzyme to use?** Multiply the desired activity by the final volume to get the total units, then divide by the specific activity of the stock: 5 U/mL × 25 mL = 125 U, and 125 U ÷ 40 U/mg = 3.125 mg. **What is one enzyme unit?** The amount of enzyme that converts 1 µmol of substrate per minute under the stated assay conditions (temperature, pH, substrate concentration). **How do you convert units to katal?** 1 katal = 1 mol/s = 6 × 10⁷ U, so 1 U = 16.67 nkat and 1 nkat = 0.06 U. **What is specific activity?** Activity per mass of protein, usually U/mg. It measures purity: it rises during purification and is highest for the pure enzyme. **How do you calculate enzyme activity from absorbance?** Divide ΔA/min by ε × path length to get M/min, multiply by the assay volume (in mL) × 1000 to get µmol/min, divide by the enzyme volume in mL and multiply by any dilution factor. ΔA/min = 0.12 with NADH (ε = 6220), 1 mL assay, 20 µL of a 1 : 10 dilution gives 9.65 U/mL. **What molar absorptivity should I use for NADH?** 6 220 M⁻¹ cm⁻¹ at 340 nm (6.22 mM⁻¹ cm⁻¹), the same for NADPH. p-Nitrophenol at 405 nm under alkaline conditions is about 18 000 M⁻¹ cm⁻¹, but check the value for your exact buffer and pH. **Why does activity depend on temperature and pH?** Rates roughly double for every 10 °C rise until the enzyme denatures, and each enzyme has a pH optimum. A unit is only meaningful together with its assay conditions. **Why should I use only the linear part of the absorbance trace?** The initial rate reflects the enzyme amount; later the substrate runs out or product inhibits the reaction and the slope falls, underestimating activity. ## Sources - [IUBMB – Enzyme nomenclature and units](https://iubmb.qmul.ac.uk/enzyme/) - [IUPAC Compendium of Chemical Terminology (Gold Book)](https://goldbook.iupac.org/) - [OpenStax – Biology 2e](https://openstax.org/details/books/biology-2e)