Enzyme Activity Calculator (U/mL and specific activity from ΔA/min)

Understanding the enzyme activity calculator — concept and principle

This calculator turns the slope of a spectrophotometer reading, the change in absorbance per minute, into enzyme activity in units per millilitre, and into specific activity per milligram of protein. It is used in biochemistry labs for kinetic assays, especially NADH- or NADPH-linked assays read at 340 nm.

Beer–Lambert turns the absorbance slope into µmol per minute; volumes and dilution scale it to the stock.

How it is calculated

Δc per minute = (ΔA/min) ÷ (ε × l)

Activity in the cuvette (U) = Δc × V_total

Stock activity (U/mL) = (ΔA/min × V_total × DF) ÷ (ε × l × V_enzyme)

Specific activity (U/mg) = (U/mL) ÷ (mg protein/mL)

ε is the molar absorption coefficient in mM⁻¹ cm⁻¹, l the path length in cm, volumes in mL, DF the dilution factor of the enzyme sample.
1 U = 1 µmol of substrate converted per minute = 16.67 nkat.

The principle

The Beer–Lambert law says absorbance is proportional to concentration: A = εcl. So a steady change in absorbance per minute is a steady change in the concentration of the coloured or UV-absorbing species. With ε in mM⁻¹ cm⁻¹ the rate comes out in mmol/L per minute, which is the same as µmol per mL per minute. Multiplying by the volume in the cuvette gives the amount converted per minute, and dividing by how much enzyme was added scales it back to the original sample.

Worked example

Default inputs: ΔA/min 0.150, ε 6.22 mM⁻¹ cm⁻¹ (NADH at 340 nm), l 1 cm, total volume 1.0 mL, enzyme 0.05 mL, dilution 10, protein 2 mg/mL.

Δc = 0.150 ÷ (6.22 × 1) = 0.02412 mM/min = 24.12 µmol/L per min.
Activity in the cuvette = 0.02412 × 1.0 = 0.02412 U.
Diluted sample = 0.02412 ÷ 0.05 = 0.4823 U/mL.
Stock = 0.4823 × 10 = 4.823 U/mL, which is 80.39 nkat/mL.
Specific activity = 4.823 ÷ 2 = 2.412 U/mg protein.

Each point is the calculator re-run with only “Molar absorption coefficient ε (mM⁻¹ cm⁻¹)” changed; the gold point is the default example. Change the inputs above and this chart follows your numbers.

Molar absorption coefficient ε (mM⁻¹ cm⁻¹)	Enzyme activity of the stock (U/mL)
1.55	19.29
3.11	9.646
4.67	6.431
6.22	4.823
7.78	3.859
9.33	3.215
12.4	2.412

How to read the result

Specific activity is the best measure of purity: it rises as a purification removes other proteins. Absolute U/mL is what you need to plan how much enzyme to add to a reaction. Only trust the number if the slope was taken from the straight, initial part of the trace and the ΔA/min sits in a range where the photometer is linear.

Common mistakes

Forgetting to subtract the blank (no-enzyme) slope.
Using a slope from the curved part of the trace, after substrate starts to run out.
Mixing ε units, such as M⁻¹ cm⁻¹ with an mM formula, which gives a 1,000-fold error.
Leaving out the dilution factor of the enzyme sample.

Keep in mind: Activity depends on temperature, pH, substrate concentration and buffer. Report those conditions with every value.

Frequently asked questions

What is one enzyme unit?

The amount of enzyme that converts 1 µmol of substrate per minute under the stated conditions.

What is a katal?

The SI unit of catalytic activity, 1 mol per second; 1 U equals 16.67 nkat.

Why is 6.22 used for NADH?

It is the standard molar absorption coefficient of NADH at 340 nm in mM⁻¹ cm⁻¹.

Does the sign of ΔA matter?

No. Assays that consume NADH show a falling absorbance; the size of the slope is what counts.

Sources: IUBMB Nomenclature Committee recommendations on enzyme units and the katal · B. L. Horecker and A. Kornberg, J. Biol. Chem. 175, 385 (1948)

Checked 9 Nov 2026. Enzyme unit (1 U = 1 µmol/min) and katal: IUBMB Nomenclature Committee recommendations; Beer–Lambert law A = εcl. NADH ε₃₄₀ = 6.22 mM⁻¹ cm⁻¹ is the standard literature value (Horecker & Kornberg, J. Biol. Chem. 175, 385, 1948).

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