What Is the Enzyme Activity Calculator?
This tool measures how fast an enzyme converts substrate, expressed in international units (U), where 1 U = 1 µmol of substrate transformed per minute. It applies the Beer–Lambert law to convert a measured change in absorbance into a molar amount of product and then normalizes that rate by the volume of enzyme used. It is a universal biochemistry/enzymology tool and is not specific to any country.
How to Use It
Run your spectrophotometric assay and record the absorbance change (ΔA) over a fixed time. Enter ΔA, the reaction time in minutes, the molar extinction coefficient ε of your chromophore (in mM⁻¹·cm⁻¹), the cuvette path length (usually 1 cm), the total reaction volume, and the volume of enzyme sample added. Make sure ε and your volumes share consistent units so the result comes out in U/mL.
The Formula Explained
$$\text{Activity (U/mL)} = \dfrac{\Delta A \times V_{total}}{\varepsilon \times l \times t \times V_{enzyme}}$$ The term \(\dfrac{\Delta A}{\varepsilon \times l}\) gives the change in concentration (mM) of product from Beer's law. Multiplying by \(V_{total}\) converts concentration to µmol of product. Dividing by time \(t\) gives µmol/min (units, U), and dividing by \(V_{enzyme}\) normalizes to activity per mL of enzyme.
Worked Example
Suppose \(\Delta A = 0.3\), \(t = 1\) min, \(\varepsilon = 6.22\) mM⁻¹·cm⁻¹ (NADH at 340 nm), \(l = 1\) cm, \(V_{total} = 3\) mL, and \(V_{enzyme} = 0.1\) mL. Then activity $$= \frac{0.3 \times 3}{6.22 \times 1 \times 1 \times 0.1} = \frac{0.9}{0.622} \approx 1.447 \text{ U/mL}.$$ The total cuvette rate is \(\dfrac{0.9}{6.22} \approx 0.1447\) µmol/min.
FAQ
What units should ε be in? Use mM⁻¹·cm⁻¹ together with volumes in mL to get U/mL. NADH at 340 nm is 6.22 mM⁻¹·cm⁻¹.
What is one Unit (U)? One unit is the amount of enzyme that converts 1 µmol of substrate per minute under the assay conditions.
How do I get specific activity? Divide the U/mL result by the protein concentration (mg/mL) of your enzyme sample to obtain U/mg.
