What exactly is one enzyme unit?

One international unit, or IU, is the amount of enzyme that converts one micromole of substrate per minute under defined assay conditions. All the numbers this tool reports are anchored to that definition, so activity scales directly with the reaction rate.

How does absorbance turn into a reaction rate?

The Beer-Lambert law links absorbance to concentration through the extinction coefficient and path length. Dividing the absorbance change by time gives the rate of concentration change, and multiplying by the reaction volume gives micromoles of product made per minute.

What is the difference between U/mL and U/mg?

Volume activity in U per mL describes how active a given volume of your enzyme prep is. Specific activity in U per mg divides that by protein concentration, giving activity per unit protein, which is the standard measure of enzyme purity and is what you track during purification.

Why must I measure during the linear phase?

The micromole-per-minute definition assumes a constant rate. Early in a reaction, before substrate depletion or product inhibition set in, the absorbance rises linearly. Reading the slope during that window gives an accurate initial velocity and a valid unit value.

What extinction coefficient should I use?

Use the coefficient of the species you are actually measuring at your assay wavelength, not the substrate. A classic example is NADH at 340 nm, with an extinction coefficient of 6220 per molar per centimetre, used in many dehydrogenase assays.

Enzyme Unit Activity Calculator

A spectrophotometric enzyme assay watches a coloured or UV-absorbing product accumulate over time. This calculator turns that absorbance slope into proper international units, then into volume activity and specific activity, using the Beer-Lambert law and the definition of an enzyme unit.

How it works

By Beer-Lambert, A = eps x c x l, so a measured absorbance change corresponds to a concentration change of deltaA / (eps x l). Dividing by the assay time gives the rate of concentration change per minute.

Multiplying that rate by the reaction volume converts it to micromoles of product per minute, which is exactly the international unit. Dividing by the enzyme volume added gives U/mL, applying any dilution factor recovers the stock activity, and dividing by protein concentration gives specific activity in U/mg.

Worked example

An assay produces NADH measured at 340 nm with an extinction coefficient of 6220, a 1 cm cuvette, and a 1 mL reaction. A change of 0.30 absorbance units in one minute is 0.30 / (6220 x 1) = 4.82e-5 mol/L/min, or about 0.048 µmol/min, which is 0.048 IU in the cuvette. With 0.05 mL of enzyme added that is about 0.96 U/mL.

Tips

Always use a blank to subtract non-enzymatic drift and read the slope only over the linear region. Confirm your extinction coefficient matches your wavelength and buffer. For coupled assays, the coefficient is that of the final reporter species, not the original substrate.