Enzyme calculations - enzyme activity and specific activity

Summary

This video tutorial explains how to calculate enzyme activity, a common challenge for students due to unit tracking errors. The process is broken down into three steps: measuring absorbance, converting it into concentration, and determining enzyme units. The video uses an example involving a colorimetric assay and explains how absorbance is used to calculate enzyme activity in micro moles per minute. It also touches on calculating specific activity, a key measure of enzyme purity, which reflects the active enzyme relative to total protein concentration in the solution.

Takeaways

• 📏 Enzyme activity is the ability to produce product in a given amount of time, typically measured in micromoles of product formed per minute.
• 🧪 The standard unit of enzyme activity is the production of 1 micromole of product per minute, referred to as one enzyme unit.
• 🥼 In a colorimetric assay, 980 microliters of substrate is mixed with 20 microliters of enzyme, and the reaction is stopped after 5 minutes by adding a buffer.
• ⏲️ After 5 minutes, the solution's absorbance is measured, and an absorbance of 0.35 is recorded.
• 🔬 The change in absorbance per minute is calculated by dividing the total absorbance (0.35) by the reaction time (5 minutes), yielding 0.07 per minute.
• 📚 Using the Beer-Lambert law, absorbance is converted to concentration by dividing the absorbance by the absorption coefficient (4600 L·mol⁻¹·cm⁻¹) and path length (1 cm).
• 🧮 The enzyme produces 1.52 × 10⁻⁵ moles per liter per minute, equivalent to 15.2 micromoles per liter per minute, which is further scaled to total enzyme volume.
• ⚖️ The total enzyme activity is 0.609 micromoles per minute for 20 microliters of enzyme, converted to 3.04 micromoles per minute per milliliter of enzyme.
• 🔍 Specific activity is a measure of enzyme purity, calculated by dividing enzyme activity per milliliter by the protein concentration (e.g., 10 mg/mL).
• 🔑 The specific activity of the enzyme solution in this example is 0.304 micromoles per minute per milligram of protein, indicating enzyme purity.

Q & A

What is enzyme activity?

Enzyme activity is the ability of an enzyme to produce a product in a specific amount of time. It is typically measured in micromoles of product formed per minute.

What is one unit of enzyme activity?

One unit of enzyme activity is defined as the amount of enzyme that produces 1 micromole of product per minute under specific conditions.

What is the significance of the Beer-Lambert Law in enzyme activity calculations?

The Beer-Lambert Law helps convert absorbance changes measured during an enzyme assay into concentration changes, which are crucial for determining enzyme activity.

How is absorbance converted to concentration using the Beer-Lambert Law?

Absorbance (A) is related to concentration (C) by the equation A = εCL, where ε is the molar absorption coefficient and L is the path length. To find concentration, divide the absorbance by the product of ε and L.

How do you calculate the concentration change per minute in an enzyme assay?

First, determine the absorbance change per minute (ΔA/min) by dividing the total absorbance change by the time. Then, use the Beer-Lambert Law to convert absorbance to concentration.

What is the process for calculating enzyme units from a given absorbance change?

First, calculate the absorbance change per minute. Then, convert it into concentration using the Beer-Lambert Law. Finally, multiply the concentration by the total volume to get micromoles of product formed per minute.

How do you calculate the enzyme activity per milliliter of enzyme solution?

To calculate enzyme activity per milliliter, divide the total activity in micromoles per minute by the volume of enzyme solution used, expressed in milliliters.

What is specific activity, and how is it calculated?

Specific activity is a measure of enzyme purity. It is calculated by dividing the enzyme activity (in micromoles per minute per milliliter) by the concentration of protein (in milligrams per milliliter).

Why is specific activity important in enzyme assays?

Specific activity indicates how much of the enzyme is active relative to other proteins in the solution. It helps assess enzyme purity and the effectiveness of purification processes.

What does the absorbance change (ΔA) of 0.35 represent in the enzyme assay described?

The absorbance change of 0.35 represents the amount of product formed by the enzyme in 5 minutes, measured through a spectrophotometer.