Enzyme Activity Calculator (U, U/mg and katal)

Background.

An enzyme is almost never weighed. It is counted by what it does. One unit, U, is the quantity of enzyme that converts one micromole of substrate per minute under stated conditions, the definition fixed by the International Union of Biochemistry's Commission on Enzymes in 1961. The SI version arrived in 1999, when Resolution 12 of the General Conference on Weights and Measures gave mole per second the name katal for catalytic activity. Since a micromole per minute is 10⁻⁶ mol spread over 60 s, one unit is exactly 50/3 nanokatals, about 16.67.

Two kinds of measurement can be converted. A continuous spectrophotometric assay gives a slope, absorbance change per minute, which Beer–Lambert turns into a rate of concentration change. A stopped assay, an HPLC run or a titration gives an amount converted over a known time, which is already a rate once divided by the minutes. Either way you get the activity in the reaction vessel, the activity per millilitre of your enzyme preparation, the specific activity per milligram of protein, and the same figures in katals.

What is enzyme activity calculator?

Catalytic activity is the rate of conversion an enzyme brings about in a defined assay, which makes it measurable even when the enzyme's purity and molar concentration are unknown. Three related figures come out of it. Activity per millilitre of preparation, which IUPAC calls catalytic-activity concentration, says how much enzyme is in the tube. Activity per milligram of protein, catalytic-activity content or specific activity, says how pure it is. Total activity, units per millilitre times the volume you hold, says how much you have.

How to use this calculator.

1. 01Pick the method: absorbance mode for a continuous rate from a spectrophotometer or plate reader, direct mode when you know the micromoles of substrate used up or product formed over a fixed time.
2. 02In absorbance mode, enter the slope of the straight, early part of the trace as a positive number, even when the absorbance falls as NADH is oxidised. Add the extinction coefficient your method names and the path length: 1 cm for a standard cuvette, a measured figure for a plate well.
3. 03Enter the total reaction volume, enzyme included, and the chromophore-per-substrate factor: 1 when you watch the reaction itself, 2 when a coupled system makes two NADH per substrate converted.
4. 04In direct mode, enter the micromoles converted and the minutes, keeping the time short enough that the rate held steady.
5. 05Enter the volume of enzyme solution added and its protein concentration from a Bradford, BCA or A280 reading.

The formula.

Beer–Lambert says absorbance A = ε·c·l, where ε is the molar extinction coefficient in M⁻¹cm⁻¹, c the concentration in mol/L and l the path length in cm. Differentiating with respect to time gives the rate of concentration change: dc/dt = (ΔA/min) ÷ (ε × l), in mol per litre per minute. Multiplying by the reaction volume in litres gives moles per minute, and multiplying by 10⁶ gives micromoles per minute, which is units. Folding the volume conversions together:

U in the reaction = ΔA/min × V_total (mL) × 1000 ÷ (ε × l × s)

The stoichiometric factor s divides because the instrument sees the chromophore, while the unit is defined on substrate: if two NADH appear per substrate molecule, the absorbance moves twice as fast as the substrate is used. Dividing by the enzyme volume added gives U/mL of the preparation, and dividing that by protein concentration gives U/mg, with the millilitres cancelling.

The SI figures need no measurement. One U is 10⁻⁶ mol per 60 s, so U/mL × 50/3 gives nkat/mL, and U/mg ÷ 60 gives kat/kg. In direct mode Beer–Lambert drops out entirely: units in the reaction are simply micromoles converted ÷ minutes.

A worked example.

Frequently asked questions.

References& sources.

1. [1]Dybkær, R. (2001). 'Unit "katal" for catalytic activity (IUPAC Technical Report).' Pure and Applied Chemistry 73(6): 927–931. DOI 10.1351/pac200173060927. Gives the katal definition (kat = 1 mol·s⁻¹), the IUB 1961 enzyme unit (U = 1 µmol·min⁻¹), the conversion '1 U = 1 µmol·min⁻¹ ≈ 16.67 nkat', CGPM Resolution 12 (1999), and the derived-quantity table used for this page's outputs: catalytic-activity concentration b = z/V (kat/m³) and catalytic-activity content z/m (kat/kg). Read directly from the IUPAC PDF, 2026-07-29. Free.
2. [2]Bureau International des Poids et Mesures, The International System of Units (SI), 9th edition (2019). Table 4 lists catalytic activity — katal — kat — mol s⁻¹ among the SI derived units with special names; Table 5 lists catalytic activity concentration as kat m⁻³. Appendix 1 reproduces Resolution 12 of the 21st CGPM (1999) verbatim, including the statement that U equals 1 µmol·min⁻¹ and the requirement that 'the measurement procedure must identify the indicator reaction'. Independent second authority; agrees with the IUPAC report on every point. Read directly from the BIPM PDF, 2026-07-29. Free.
3. [3]Horecker, B.L. & Kornberg, A. (1948). 'The extinction coefficients of the reduced band of pyridine nucleotides.' Journal of Biological Chemistry 175: 385–390. PMID 18873313. The original determination of the 340 nm extinction coefficient of reduced pyridine nucleotides, undertaken because published values 'showed considerable variation', and the source of this page's 6220 M⁻¹cm⁻¹ default. The competing value of 6300 M⁻¹cm⁻¹ found elsewhere in the literature differs by 1.3%, which is why ε is an editable input here rather than a constant. Publisher-hosted full text; PubMed record open.
4. [4]International Union of Biochemistry, Report of the Commission on Enzymes (Pergamon Press, Oxford, 1961). The original definition of the enzyme unit as the amount catalysing the conversion of 1 µmol of substrate per minute under specified conditions. PRINT / BIBLIOGRAPHIC ONLY — not available online; its content is quoted here through the IUPAC and BIPM references above, both of which reproduce the definition verbatim.
5. [5]Nomenclature Committee of the International Union of Biochemistry (1979). 'Units of Enzyme Activity.' European Journal of Biochemistry 97(2): 319–320. DOI 10.1111/j.1432-1033.1979.tb13116.x. The IUB nomenclature committee's own statement of the relationship between the enzyme unit and the katal. PAYWALLED — the publisher returned a payment-required response on retrieval (2026-07-29), so this is cited bibliographically; its content is confirmed through the IUPAC and BIPM references above.