0.1
AU/min
0.0482
µmol/min
0.4823
U/mL
0.0482
U
0.0161
mM/min
0.1
AU/min
0.0482
µmol/min
0.4823
U/mL
0.0482
U
0.0161
mM/min
The Enzyme Activity Calculator determines the catalytic activity of an enzyme preparation from spectrophotometric assay data. Enzyme activity is measured in International Units (U), where one unit converts one micromole of substrate per minute under specified conditions. This is the standard way to quantify how much active enzyme is present in a sample.
By entering the change in absorbance, assay conditions, and volumes, you can calculate the activity per milliliter of enzyme solution. This calculation is fundamental in enzymology for enzyme purification, quality control of enzyme preparations, and comparing activity across different enzyme sources or batches.
Enzyme activity is calculated using Beer-Lambert Law to convert absorbance changes to concentration changes:
Activity (U/mL) = (ΔA × Total Volume) / (ε × l × Enzyme Volume × Time)
Where ΔA is the absorbance change, ε is the molar extinction coefficient (in mM⁻¹cm⁻¹), l is the path length (cm), and time is in minutes. The result is in µmol/min/mL = U/mL. Total activity is the activity per mL multiplied by the enzyme volume.
Inputs
Results
Using the NADH extinction coefficient of 6.22 mM⁻¹cm⁻¹ at 340 nm, an absorbance change of 0.5 in 5 minutes gives 0.48 U/mL enzyme activity.
Inputs
Results
With the p-nitrophenol extinction coefficient at 405 nm, 0.05 mL of enzyme producing ΔA of 1.2 in 10 minutes has activity of 0.26 U/mL.
One International Unit (U) is the amount of enzyme that catalyzes the conversion of one micromole of substrate per minute under defined conditions of temperature, pH, and substrate concentration. The SI unit katal (mol/s) is also used: 1 U = 16.67 nanokatal.
The extinction coefficient converts absorbance changes to concentration changes via Beer-Lambert Law (A = εlc). Common values include 6.22 mM⁻¹cm⁻¹ for NADH at 340 nm and 18.3 mM⁻¹cm⁻¹ for p-nitrophenol at 405 nm. Using the wrong coefficient gives incorrect activity values.
Initial velocity requires that the absorbance change is linear with time, meaning less than 10% of substrate has been consumed. Use the linear portion of your progress curve and ensure the reaction rate is constant over your measurement interval. Non-linear progress indicates substrate depletion or product inhibition.
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