Enzyme calculations - enzyme activity and specific activity

Gus Cameron
21 Oct 201810:39

Summary

TLDRThis 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.

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Ähnliche Tags
Enzyme ActivityBiochemistryLab AssayAbsorbanceBeer-Lambert LawSpecific ActivityConcentrationMicromolesProtein PurityColorimetric Assay
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