Enzyme Activity / Specific Activity Calculator

Calculate total enzyme activity in Units (U), volume activity (U/mL), and specific activity (U/mg protein) from spectrophotometric rate measurements.

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Assay Parameters

Enter spectrophotometer absorbance slope, extinction coefficient, and volumes.

Absorbance Change Rate (ΔA / min)

Extinction Coefficient ε (mM⁻¹ cm⁻¹)

Pathlength of Cuvette (cm)

Total Assay Volume (mL)

Enzyme Sample Volume added (mL)

Protein Concentration of Sample (mg/mL)

Volumetric Activity

0.0804 U/mL

Specific Activity

0.1608 U/mg

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Definition of Enzyme Unit (U)

1 Unit (U) = 1 μmol substrate converted per minute. Katals (kat) represent 1 mol/s (1 U = 16.67 nkat).

Common Chromogenic Substrate Extinction Coefficients Reference

Cofactor / Chromophore	Wavelength (λ)	Extinction Coeff ε (M⁻¹ cm⁻¹)	Extinction Coeff ε (mM⁻¹ cm⁻¹)
NADH / NADPH (Oxidoreductases)	340 nm	6,220 M⁻¹ cm⁻¹	6.22 mM⁻¹ cm⁻¹
p-Nitrophenol (pNPP / Alkaline Phosphatase)	405 nm	18,500 M⁻¹ cm⁻¹	18.5 mM⁻¹ cm⁻¹
o-Nitrophenol (ONPG / β-Galactosidase)	420 nm	4,500 M⁻¹ cm⁻¹	4.50 mM⁻¹ cm⁻¹
Ferricyanide (Electron acceptors)	420 nm	1,000 M⁻¹ cm⁻¹	1.00 mM⁻¹ cm⁻¹

Calculation Methodology & Equations

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Volumetric Activity (U/mL)

Vol Activity (U/mL) = (ΔA/min × V_assay) / (ε × d × V_sample)

Where ΔA/min is absorbance slope, V_assay is total reaction volume (mL), ε is extinction coefficient (mM⁻¹ cm⁻¹), d is pathlength (cm), and V_sample is added enzyme volume (mL).

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Specific Activity (U/mg)

Specific Activity (U/mg) = Volumetric Activity (U/mL) / Protein Conc (mg/mL)

Measures enzyme purity. Specific activity increases during purification steps as non-target proteins are removed.

Frequently Asked Questions

What is the difference between Enzyme Activity (U/mL) and Specific Activity (U/mg)? ▼

Enzyme Activity (volumetric activity, U/mL) measures the catalytic units per milliliter of sample. Specific Activity (U/mg) divides volumetric activity by total protein concentration (mg/mL), reflecting enzyme purity.

How is an Enzyme Unit (U) defined? ▼

One International Unit (U) of enzyme activity is defined as the amount of enzyme that catalyzes the transformation of 1 micromole (μmol) of substrate per minute under specified assay conditions.

What unit should be used for the extinction coefficient ε? ▼

Enter the extinction coefficient in mM⁻¹ cm⁻¹. If your extinction coefficient is provided in M⁻¹ cm⁻¹, divide the value by 1,000 before entering.