Enzyme Specific Activity Calculator

Enzyme units, volumetric activity and specific activity (U/mg) from absorbance change.

Activity (U/mL enzyme) = (ΔA/min × V_total) ÷ (ε × l × V_enzyme) × dilution, with ε in mM⁻¹cm⁻¹ and volumes in mL. Specific activity = U/mL ÷ protein (mg/mL). One unit (U) converts 1 µmol of substrate per minute.

Free, runs in your browser. Nothing you enter is uploaded.

How to use it

  1. Choose absorbance (ΔA/min) or µmol of product.
  2. Enter ΔA/min, the extinction coefficient, path length, reaction and enzyme volumes, dilution and protein concentration.
  3. Read units in the cuvette, U/mL and specific activity in U/mg.

What you enter

What you get

Formula

U/mL = (ΔA/min × V_total) ÷ (ε × l × V_enzyme) × DF | Specific activity = (U/mL) ÷ [protein mg/mL]

Worked example

Input: ΔA340 = 0.124/min, NADH, 1 cm, 1.0 mL total, 0.02 mL enzyme, DF 10, 2 mg/mL protein

Result: ≈ 9.97 U/mL, 4.98 U/mg

Each mg of protein converts about 5 µmol of NADH per minute.

Questions

How do you calculate enzyme specific activity?

Find the enzyme activity in units (µmol/min) and divide by the mg of protein in the assay. It is reported as U/mg.

What is the difference between activity and specific activity?

Activity is the total rate (U or U/mL). Specific activity divides by protein, so it rises as a purification removes other proteins and shows purity.

What extinction coefficient should I use for NADH?

6.22 mM⁻¹cm⁻¹ at 340 nm. p-Nitrophenol is about 18.3 mM⁻¹cm⁻¹ at 405 nm in alkaline buffer, and TNB from DTNB is 14.15 mM⁻¹cm⁻¹ at 412 nm.

What is a katal?

The SI unit of enzyme activity: 1 mol/s. 1 U = 16.67 nkat.

Limitations

References

Related reading

More Molecular Assays

All free tools · By Dr. Zubair Khalid, DVM, MS, PhD.