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4: Activity 1-4 - Analysis of Enzymatic Activities of Cell-Free Extracts

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    Learning Objectives
    • Understand the role of cytochrome P450 enzymes in biological systems.
    • Explain the importance of NADPH in P450-catalyzed hydroxylation reactions.
    • Define and apply Beer’s Law in the context of spectrophotometric analysis.
    • Describe how absorbance changes at 340 nm are used to track enzyme activity.
    • Prepare a reaction mixture and outline the rationale behind each component.
    Definition: Term
    • Cytochrome P450: A family of heme-containing enzymes involved in oxidation reactions, often used to metabolize drugs and fatty acids.
    • P450BM3: A bacterial version of P450 that is soluble and highly active; often used in research for enzymatic activity studies.
    • NADPH (Nicotinamide Adenine Dinucleotide Phosphate, Reduced): A cofactor that donates electrons in biosynthetic reactions; oxidized to NADP⁺ in P450 activity.
    • Beer’s Law: A relationship that connects absorbance (A) to the concentration (c) of a substance in solution: A = εcl.
    • Extinction Coefficient (ε): A constant that indicates how strongly a substance absorbs light at a particular wavelength. For NADPH at 340 nm, ε = 6,220 M⁻¹cm⁻¹.
    • Specific Activity: A measure of enzyme activity normalized to protein concentration: µmol of substrate converted per second per mg protein.
    • Spectrophotometer: An instrument that measures how much light a sample absorbs at a particular wavelength.
    • Absorbance at 340 nm: A measure used to monitor NADPH oxidation in real time. As NADPH is used up, absorbance decreases.
    Pre-Lab Questions
    1. What role does NADPH play in P450-mediated reactions?
    2. Why is 340 nm specifically chosen for measuring NADPH oxidation?
    3. What is the significance of a blank sample in spectrophotometric experiments?
    4. Why do we incubate the mixtures at 37°C before adding NADPH?
    5. What would it mean if there’s no change in absorbance over time?

    Beer's Law Spectrophotometric Analysis of P450 Enzyme Expression

    In this activity, we will analyze the enzymatic activity of cell-free extracts that contain the cytochrome P450BM3 enzyme. Cytochrome P450 enzymes are a large family of heme-containing monooxygenases that play critical roles in the metabolism of a wide range of endogenous and exogenous compounds. One of the most common ways to measure the activity of these enzymes is by monitoring the oxidation of NADPH (nicotinamide adenine dinucleotide phosphate, reduced form), which acts as an essential cofactor in the P450 catalytic cycle.

    The enzymatic activity can be assessed by measuring the change in absorbance at 340 nm using a spectrophotometer. This wavelength corresponds to the absorbance peak of NADPH, which decreases as it is oxidized to NADP⁺ during the enzymatic reaction. Specifically, for each hydroxylation reaction catalyzed by cytochrome P450BM3, one molecule of NADPH is consumed. Therefore, the rate of NADPH oxidation provides a direct and quantitative measure of the enzyme’s catalytic activity. The molar extinction coefficient of NADPH at 340 nm is 6,220 M⁻¹cm⁻¹, which is used to convert absorbance changes into actual concentrations and reaction rates. This spectrophotometric method provides a reliable, real-time approach to evaluate the activity of cytochrome P450 enzymes in biological samples.

    Materials:

    1. Cell-free extracts (CFE) 
    2. 1.5 mL centrifuge tubes
    3. Phosphate buffer (PO4 buffer): 100 mM potassium phosphate, pH 7.4 (Pre-incubated at 37°C), OR 0.25X PBS
      • Made from 1L of a 1M stock solution with the following:
        • 95g of monobasic potassium phosphate (Carolina, #884250)
        • 52.5g of dibasic potassium phosphate (Carolina, #884300)
    4. Distilled water
    5. 10 mM lauric acid in 50 mM potassium carbonate (stored at -20°C)
      • Lauric acid (Carolina, #871840)
      • Potassium carbonate (Carolina, #882836)
      • Note: Make sure to filter the Lauric acid!
    6. 10 mM NADPH in water (stored at -20°C)
      • Always make this fresh. This can only last up to 3 months. 
    7. UV/VIS Spectrophotometer (Thermo Scientific, #840-300000)
    8. UVette cuvettes (Eppendorf, #952010051)
    9. Pipettes and tips
    10. Parafilm, Sharpie, acetone, cotton, scissors

    Procedure:

    1.  Pre-warm the following tubes at 37°C.
      • Cell-Free Extract (CFE)
        • Only take an aliquot of CFE, do not warm all of them as they will degrade over time if not frozen!
      • Phosphate buffer
      • Lauric Acid
    2. Obtain a cuvette and mix the following components (refer to the table below). Do not add NADPH yet.
      • 50 μL of 10 mM Lauric acid
      • 390 μL of Phosphate buffer
      • 50 μL of CFE
    3. Blank the spectrophotometer using this mixture (From step 2) at 340 nm .
    4. Add 10 μL of NADPH to the reaction mixture and seal it with parafilm.
    5. Quickly mix the cuvette and immediately measure the initial absorbance at 0 seconds and the final absorbance at 2 minutes.
    6. You may need to repeat this several times: Optimize the volume of Lauric acid, CFE, and/or NADPH to achieve a decrease in absorbance between 0.2 to 0.4 after 2 minutes.
      • Increase lauric acid concentration (affects km): Providing more substrate can enhance enzyme turnover, leading to a faster NADPH oxidation and greater absorbance drop.
      • Increase enzyme (CFE) volume (affects Vmax): More enzyme increases catalytic capacity, accelerating the reaction rate.

    Measuring P450-Driven NADPH Oxidation:

    1.  Record the Absorbance at two time points
      • For example
        • 0 second = 0.4192
        • 120 second = 0.2451
    2. Calculate the differences in the two absorbance values
      • For example: 0.4192 – 0.2451 = 0.1741
    3. Convert Absorbance Change to NADPH Concentration (extinction coefficient is 6220 M⁻¹cm⁻¹).
      • c = A/EL
        • Δ NADPH concentration=
    4. Calculate the initial rate
      • rate = Δ concentration / Δtime
        • 28 μM / 120 s = 0.233 μM/s
    5. Determine Protein Amount in the Reaction
      • Use Bradford assay to get protein concentration (e.g., 0.119 mg/mL)
      • Multiply by volume added (e.g., 0.1 mL)
        • Protein amount = 0.119mg/mL × 0.1mL = 0.0119mg
    6. Normalize Rate to Protein
      • Specific activity = rate / protein 
        • (0.233 μM/s) / (0.0119mg) = 19.6μM/s/mg

     

    Specific Activity (µM/s/mg)

    Rating

    Interpretation

    >15

    ⭐ Excellent

    High activity; Deserves Extra credit!

    5–15

    ✅ Good

    Useful enzyme.

    1–5

    ⚠️ Moderate

    Low quality.

    <0.1

    🚫 Very Poor

    Dead protein.

     

    clipboard_ea5c4ef412a8b4894317c0686bf3a38fc.png

    Image of a flow chart summarizing the Analysis of Enzymatic Activities of Cell-Free Extracts. Image created by Diana Valdovinos. 

    Post-Experiment Questions:

    1. Graph the Absorbance vs. Time:
      • Create a graph of absorbance over time.
      • Add a trendline to the data.
      • The slope of the trendline will give the rate of the reaction in terms of optical density (O.D.) per second.
    2. Calculate the Rate of NADPH Oxidation:
      • Use the following equation, derived from Beer’s law: O.D.=E×c×lO.D. = E \times c \times lO.D.=E×c×l
        • Where:
          • E is the molar extinction coefficient of NADPH at 340 nm (6220 M⁻¹cm⁻¹).
          • c is the concentration of NADPH.
          • l is the path length (in cm), typically 1 cm for cuvettes.
      • Calculate the rate of NADPH oxidation in terms of micromoles of NADPH oxidized per second.
    3. Determine the Specific Activity:
      • To calculate the specific activity of your enzyme, express the rate of NADPH oxidation in micromoles of NADPH oxidized per second per milligram of protein.
      • First, determine the amount of protein in your samples.
      • Then, calculate how much 1 mg of protein contributes to the NADPH oxidation rate.
    Learning Objectives

    By the end of this lab and lecture discussion, students should be able to:

    • Graph and interpret real-time enzymatic data.
    • Use absorbance values to calculate NADPH oxidation rates.
    • Apply Beer’s Law to convert absorbance into concentration units.
    • Determine the specific enzymatic activity of cell-free extracts.
    • Troubleshoot potential problems in enzyme assays.
    Post-Experiment Questions
    1. Graph Analysis: Use the absorbance readings you collected at 340 nm.
      • Plot Absorbance (y-axis) against Time in seconds (x-axis).
      • Add a trendline (straight line that best fits your data).
      • Find the slope of this trendline — this tells you how fast the reaction is happening, measured in Absorbance units per second (O.D./sec).
      • What does the slope of your absorbance vs. time graph tell you?
      • How did the reaction rates differ between CFE-A and CFE-B?
      • What could explain a slow or inactive sample?
    2. Beer’s Law Calculations: Now that you know how quickly the absorbance is changing, you can figure out how fast NADPH is being used up by the enzyme.
      • Use Beer’s Law: A=εcl OR c=A/(εl)
        • A is the change in absorbance
        • ε (epsilon) is the extinction coefficient of NADPH at 340 nm = 6220 M⁻¹cm⁻¹
        • c is the concentration of NADPH (what we want to find!)
        • l is the path length of the cuvette = 1 cm
      • This gives you the amount of NADPH oxidized per second in micromoles per second (µmol/sec).
      • Were your calculated NADPH oxidation rates consistent with your observations?
      • How did you use the extinction coefficient in your calculations?
    3. Specific Activity: Now let’s figure out how efficient your enzyme is based on how much protein you used.
      • First, find how many milligrams (mg) of protein were in your reaction (from earlier Bradford assay or known concentrations).
      • Then, divide your NADPH oxidation rate by the amount of protein used.
      • Final formula: Specific Activity = µmol of NADPH oxidized / second / mg protein
        • This tells you how active your enzyme is for each milligram of protein — a key measure in enzyme analysis!
      • Why is it important to normalize enzyme activity to protein content?
      • How would contamination or protein degradation affect your results?
    4. Experimental Design:
      • If you were to repeat this experiment, what would you change?
      • How could you modify the assay to test a different substrate or enzyme?

    4: Activity 1-4 - Analysis of Enzymatic Activities of Cell-Free Extracts is shared under a not declared license and was authored, remixed, and/or curated by LibreTexts.