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

Background.

An enzyme activity calculator converts what you actually measured — a slope on a spectrophotometer trace, or an amount of substrate that disappeared over a fixed time — into the standard currencies of enzymology: enzyme units per millilitre, specific activity in units per milligram of protein, and the SI equivalents in katals.

The base definition has not changed since 1961. The International Union of Biochemistry's Report of the Commission on Enzymes defined one unit, U, as the amount of enzyme catalysing the conversion of one micromole of substrate per minute under specified assay conditions. The SI arrived much later: the 21st General Conference on Weights and Measures adopted the katal in 1999, by Resolution 12, as the special name for mole per second when used to express catalytic activity. The conversion follows from the definitions alone — one micromole per minute is 10⁻⁶ mol divided by 60 s, so 1 U is exactly 50/3 = 16.67 nkat, and 1 U/mg is exactly 1/60 kat/kg. This calculator carries those as exact fractions rather than as the rounded 16.67 printed in the IUPAC technical report, which would introduce a systematic 0.02% error in every katal output.

For a continuous spectrophotometric assay the arithmetic runs through Beer–Lambert. A slope of ΔA per minute divided by ε × l gives a rate of concentration change in mol per litre per minute; multiplying by the reaction volume gives moles per minute; converting to micromoles gives units. Written out: U in the assay = ΔA/min × V_total(mL) × 1000 ÷ (ε × l × s), where s is the stoichiometric factor. Divide by the volume of enzyme you added and you have U/mL of your preparation.

That stoichiometric factor deserves attention before you use the result, not after. It is the number of moles of the molecule you are watching per mole of substrate actually converted. It is 1 when the reaction you monitor is the reaction you care about, and 2 for a coupled assay that produces two NADH per substrate turn. Getting it wrong is a silent factor-of-two error that no amount of replication will reveal, because the assay is perfectly reproducible while being twice the true value.

The extinction coefficient carries an honest disagreement, and it belongs here rather than in a footnote. The default of 6220 M⁻¹cm⁻¹ for NADH at 340 nm comes from Horecker and Kornberg's 1948 determination in the Journal of Biological Chemistry — a paper written precisely because the published values at the time 'showed considerable variation'. A substantial minority of the literature uses 6300 instead. The two differ by 1.3%, and because activity is inversely proportional to ε that disagreement propagates directly into your answer. This calculator does not pick a winner: ε is an editable field with its authority named, so you can enter whatever your method specifies. That is the only defensible treatment, because ε in any case depends on wavelength, pH, temperature, buffer and instrument bandwidth.

Two other approximations limit the result. The first is zero-order kinetics — the IUPAC technical report frames the whole measurement as one taken 'preferably so that zero-order kinetics is achieved by a much higher amount-of-substance concentration of substrate than of catalyst, giving a constant rate of conversion'. That holds only while substrate is in large excess; once it depletes the trace curves and a slope fitted across the curved region under-reports the activity. Use the initial linear region only. The second is Beer–Lambert linearity itself, which fails above roughly A = 1.5 on most instruments and in turbid or scattering samples.

Finally, the thing that most often makes a published activity number useless: an enzyme unit is defined only relative to its assay conditions. CGPM Resolution 12 says so in normative language — 'the measurement procedure must identify the indicator reaction'. Temperature, pH, buffer, substrate concentration and wavelength all change the number, and an activity measured at 25 °C is not comparable with one measured at 37 °C. This calculator returns those requirements alongside the result rather than assuming you will remember them.

What is enzyme activity calculator?

Catalytic activity is a property of a catalyst measured by the rate of conversion it catalyses, in a specified measurement system. Because it is defined by an effect rather than by an amount, it is the practical way to quantify an enzyme whose molar concentration and purity you do not know — which is nearly always the case for a crude or partly purified preparation.

The enzyme unit, U, is the traditional measure: one U is the amount of enzyme converting one micromole of substrate per minute under specified conditions. The katal is the coherent SI unit, one mole per second, adopted for catalytic activity by the 21st CGPM in 1999 after decades of advocacy from IUPAC, IFCC, IUBMB and the WHO. The katal is enormous relative to laboratory reality — one katal would convert a mole of substrate every second — so real values appear as nanokatals or microkatals, which is part of why the older unit persists in bench practice.

Three derived quantities matter, and IUPAC names them precisely. Catalytic-activity concentration is activity per unit volume of the preparation (U/mL, or kat/m³ in SI), and it answers 'how much enzyme is in this tube'. Catalytic-activity content is activity per unit mass of protein (U/mg, or kat/kg in SI) and is what everyone calls specific activity. The distinction between the two is the whole logic of a purification table: volumetric activity can be raised trivially by concentrating a sample, whereas specific activity rises only when non-enzyme protein is removed, so specific activity is the column that tells you whether a purification step worked. Total activity — units per millilitre times the volume you have — is the column that tells you what it cost in yield.

A fourth quantity, molar catalytic activity or turnover number, requires knowing the molar concentration of pure enzyme and is therefore out of reach for the crude preparations this calculator is aimed at.

The deliberately vague phrase 'under specified conditions' is doing essential work in every one of these definitions. An enzyme unit is not an intrinsic property of a protein; it is the output of a measurement procedure. Change the temperature, the pH, the buffer, the substrate concentration or the coupling system, and the number changes. This is why comparing activity values across papers is unreliable unless the assay conditions match, and why the CGPM's own resolution insists that a katal value identify the indicator reaction it came from.

How to use this calculator.

1. 01Run the assay so that the rate is genuinely initial and linear: substrate in large excess, and a slope fitted only to the straight portion of the trace. Both the unit definition and the Beer–Lambert step assume zero-order kinetics.
2. 02For a spectrophotometric assay, read the slope in absorbance units per minute and enter its magnitude — positive, even when the chromophore is being consumed and the trace falls.
3. 03Enter the extinction coefficient your method specifies. The default is 6220 M⁻¹cm⁻¹ for NADH at 340 nm; if your protocol says 6300, use 6300, and note that the choice moves your answer by 1.3%.
4. 04Enter the path length. Use 1 cm for a standard cuvette; for a microplate, measure it on your own plate at your own fill volume rather than assuming — a 200 µL well in a flat-bottom plate is roughly 0.55 cm, not 1.
5. 05Set the chromophore-per-substrate factor. Leave it at 1 for a directly monitored reaction; set it to 2 for a coupled assay producing two NADH per substrate turn. This is the most common silent factor-of-two error in enzyme assays.
6. 06Enter the total reaction volume and the volume of enzyme solution you added. The enzyme volume must not exceed the total — the enzyme is part of the reaction volume, not additional to it.
7. 07Enter your protein concentration from Bradford, BCA or A280 to get specific activity. If you only need volumetric activity, any positive value works and you can ignore the U/mg output.
8. 08For a stopped, HPLC or titration assay, switch to the direct mode and enter the micromoles converted and the time. One unit is one micromole per minute by definition, so no extinction coefficient is involved.
9. 09Record the conditions with the result: temperature, pH, buffer, substrate concentration, wavelength and the coupling system. An activity value without them cannot be reproduced or compared.

The formula.

The spectrophotometric route is Beer–Lambert run backwards. A = ε·c·l relates absorbance to concentration, so differentiating with respect to time gives dc/dt = (dA/dt) ÷ (ε × l), a rate of concentration change in mol per litre per minute. With ΔA/min = 0.100 and ε = 6220 M⁻¹cm⁻¹ over a 1 cm path, that is 0.100 ÷ 6220 = 1.6077 × 10⁻⁵ mol L⁻¹ min⁻¹.

Multiplying by the reaction volume converts a concentration rate into an amount rate. A 3.0 mL cuvette is 0.003 L, so the reaction is converting 1.6077 × 10⁻⁵ × 0.003 = 4.8232 × 10⁻⁸ mol per minute. Since one enzyme unit is one micromole per minute, multiply by 10⁶: the cuvette contains 0.048232 U. Dividing by the 0.1 mL of enzyme solution added gives 0.48232 U/mL — exactly 300/622 — which is the volumetric activity of the preparation. Collapsing the volume conversions gives the compact form U in the assay = ΔA/min × V_total(mL) × 1000 ÷ (ε × l × s).

The stoichiometric factor s sits in the denominator because the calculator watches a chromophore but reports substrate. If the indicator reaction produces two molecules of NADH for every molecule of substrate converted, the observed absorbance rate is twice the substrate conversion rate, so dividing by 2 recovers the quantity the unit is defined on. Leaving s at 1 when it should be 2 reports exactly double the true activity, reproducibly and undetectably.

Specific activity divides volumetric activity by protein concentration, and the millilitres cancel: 0.48232 U/mL ÷ 0.5 mg/mL = 0.96463 U/mg, or 600/622. The SI forms follow from the definitions alone, with no measurement involved. One unit is 10⁻⁶ mol over 60 s, which is 1.6667 × 10⁻⁸ kat, so 0.48232 U/mL × 50/3 = 8.0386 nkat/mL. One U/mg is 10⁻⁶ mol per 60 s per 10⁻⁶ kg, which is 1/60 kat/kg, so 0.96463 ÷ 60 = 0.016077 kat/kg.

The direct mode skips Beer–Lambert entirely and applies the definition of the unit to a measured amount: 12 µmol converted in 10 minutes is 1.2 µmol per minute, which is 1.2 U in the reaction; from 0.1 mL of enzyme that is 12 U/mL, and against 0.5 mg/mL protein it is 24 U/mg, 200 nkat/mL and 0.4 kat/kg. Every one of those is a whole number, which makes this mode a clean check on the unit conversions — and the test suite uses it as exactly that, confirming that a spectrophotometric result converted into an equivalent amount and fed back through the direct mode reproduces the same activity.

Rounding stage: all arithmetic runs at full Decimal.js working precision and is rounded only when the result is returned, to twelve significant figures. Significant figures rather than decimal places, deliberately — specific activity in kat/kg for a slow enzyme in a dilute preparation can sit many decades below 1, and a fixed-decimal-place boundary would round those outputs to zero. The 1 U = 50/3 nkat conversion is carried as an exact fraction throughout and never as the rounded 16.67 that appears in print. Report two or three significant figures: the dominant uncertainty is the extinction coefficient, where two literature values disagree by 1.3%, not the arithmetic.

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.