Enzyme Activity Calculator
Calculate reaction kinetics using basic rate data or spectrophotometric absorbance ($\Delta A$).
Calculated Activity
Reaction Progress
Basic Rate Calculation
Simple rate calculation showing amount of product formed per unit time. Useful for general kinetic studies.
Understanding Enzyme Kinetics
Enzymes are the biological catalysts that power life. Measuring their "Activity" tells us how many active enzyme molecules are present. This tool helps you convert raw data—like absorbance changes from a spectrophotometer—into standardized International Units (U).
Beer-Lambert Law
Most enzyme assays rely on light absorption. The law states that Absorbance (A) is proportional to concentration.
ε = Extinction Coeff | c = Conc | l = Path Length
Calculating Units
To find Activity (U/ml) from a slope (ΔA/min):
- Convert ΔA to Concentration (Δc) using ε.
- Multiply by Total Volume (Vt) to get Total Moles.
- Divide by Sample Volume (Vs) to normalize.
Critical Factors
- TempAssays must be temp-controlled (usually 25°C or 37°C).
- pHBuffer pH must match the enzyme's optimum.
- LinearityOnly measure the linear part of the slope!
Factors Affecting Enzyme Activity
Enzyme assays are sensitive experiments. Several factors can drastically alter the calculated activity, leading to erroneous results.
- Temperature: Every enzyme has an optimum temperature (often 37°C for human enzymes). Increasing temperature generally increases reaction rate by providing kinetic energy, but exceeding the optimum causes denaturation, where the protein structure unfolds and activity drops to zero.
- pH Level: Enzymes have active sites with specific ionic charges. Deviating from the optimum pH (e.g., pH 7.4 for blood enzymes, pH 2.0 for pepsin) changes these charges, preventing substrate binding.
- Substrate Saturation: This calculator assumes you are measuring the Initial Rate (V0). This is valid only when substrate concentration is saturating (far above Km), ensuring the enzyme is working at its maximum velocity (Vmax).
Medical & Industrial Applications
Why do we calculate enzyme units? It is a critical diagnostic tool in medicine and a quality control measure in industry.
Liver Function Tests
Doctors measure ALT and AST levels in the blood. These enzymes normally reside inside liver cells. High activity in the blood indicates liver cell damage (e.g., from hepatitis or alcohol), causing enzymes to leak out.
Food Industry
Amylase and Protease activities are monitored in brewing and baking to ensure dough rises correctly and beer clarifies. Lactase activity is measured to produce lactose-free milk.
Example: Calculating NADH Oxidase Activity
Let's walk through a real lab scenario. You are testing an enzyme that oxidizes NADH. NADH absorbs light at 340 nm, but NAD+ does not. As the reaction proceeds, absorbance decreases.
Data:
- Slope (ΔA/min) = -0.150
- Total Volume (Vt) = 3.0 ml
- Sample Volume (Vs) = 0.1 ml
- Extinction Coeff (ε) = 6.22 mM⁻¹cm⁻¹
- Path Length (l) = 1.0 cm
Step 1: Calculate Total Units
Δc = ΔA / ε = 0.150 / 6.22 = 0.0241 mM/min
Total Units = Δc × Vt = 0.0241 × 3.0 = 0.0723 µmol/min
Step 2: Normalize to Sample Volume
Activity = 0.0723 U / 0.1 ml = 0.723 U/ml
Explore More Biology Tools
Frequently Asked Questions
What is an Enzyme Unit (U)?
One Enzyme Unit (U) is defined as the amount of enzyme that catalyzes the conversion of 1 micromole (1 µmol) of substrate per minute under standard conditions. The SI unit is the Katal (mol/s), but Units (U) are more commonly used in labs.
How do I calculate activity from Absorbance?
Use the Spectrophotometric Mode. The formula is derived from the Beer-Lambert Law (A = εcl).\nActivity (U/ml) = (ΔA/min × Vt × 1000) / (ε × Vs × l), where ε is the extinction coefficient and l is path length (usually 1 cm).
What is Specific Activity?
Specific Activity is the Enzyme Activity (Units) divided by the total amount of protein (mg) in the sample. It is a measure of enzyme purity. Higher specific activity indicates a purer enzyme preparation.
What corresponds to the Initial Rate ($V_0$)?
The Initial Rate ($V_0$) is the slope of the linear portion of the reaction progress curve (Product vs Time) right at the beginning. This is when substrate is in excess and the reaction is proceeding at its maximum possible speed for that enzyme concentration.
Which Extinction Coefficient should I use?
It depends on your assay. For NADH/NADPH coupled assays (340 nm), use 6220 $M^{-1}cm^{-1}$. For p-Nitrophenol (405 nm), use 18000. For DTNB/Ellman's (412 nm), use 13600.
Why do I need to correct for Path Length?
Standard cuvettes have a path length of 1 cm. However, if you are using a microplate reader, the path length depends on the volume of liquid in the well. You must correct for this, or your calculated concentration will be wrong.