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3: Activity 1-3 - Beer’s Law Spectrophotometric Analysis

Last updated

Jul 16, 2025
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- 2: Activity 1-2 - Bradford Assay – Measuring Protein Concentration
- 4: Activity 1-4 - Analysis of Enzymatic Activities of Cell-Free Extracts

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Learning Objectives

Explain the principle of Beer’s Law and how it relates absorbance to concentration.
Identify the components and functions of a spectrophotometer.
Describe the structural and functional significance of the P450_BM3 enzyme.
Understand how light absorption by heme can be used as a proxy to determine enzyme concentration.

Definition: Term

Beer’s Law (Beer-Lambert Law): A relationship describing how the absorbance of light (A) is proportional to the concentration (c) of a substance, path length (L), and its extinction coefficient (ε): A = εcL.
Absorbance (A): A unitless measurement of how much light is absorbed by a sample. Also referred to as optical density (O.D.).
Extinction Coefficient (ε): A constant that reflects how strongly a substance absorbs light at a particular wavelength. Units: M⁻¹cm⁻¹.
Path Length (L): The distance light travels through the sample in the cuvette. Usually 1 cm.
Cytochrome P450: A large family of enzymes involved in oxidation reactions, many of which metabolize drugs or steroids. They contain a heme prosthetic group.
P450_BM3: A bacterial cytochrome P450 enzyme from Bacillus megaterium that is self-sufficient and highly active, commonly used in biochemistry studies.
Heme: An iron-containing porphyrin ring that binds oxygen and gives P450 enzymes their characteristic absorbance near 418 nm.
UV/Vis Spectrophotometer: An instrument that measures how much UV or visible light a sample absorbs. Used to quantify analyte concentration.

Beer's Law Spectrophotometric Analysis of P450 Enzyme Expression

In biochemistry, the spectrophotometer is a critical tool used to analyze the concentration of molecules in solution, particularly those that absorb light at specific wavelengths. This analytical method is based on the Beer-Lambert Law (commonly referred to as Beer’s Law), which describes the relationship between the absorbance of light by a substance and its concentration in solution. The fundamental equation for Beer’s Law is expressed as:
A = εcL, where A is the absorbance (also known as optical density or O.D.), ε (epsilon) is the molar extinction coefficient (in units of M⁻¹cm⁻¹), c is the concentration of the absorbing species in moles per liter (M), and L is the path length of the light through the sample (usually in centimeters).

This law indicates that absorbance is directly proportional to both the concentration of the substance and the distance the light travels through the solution. For example, an absorbance of 1.0 corresponds to a tenfold decrease in transmitted light intensity compared to the incident light, meaning only 10% of the original light passed through the sample. The spectrophotometer measures the intensity of light before and after it passes through the solution, allowing us to determine the absorbance and, using Beer’s Law, calculate the concentration of the molecule of interest.

In this lab, you will apply Beer’s Law to measure the activity of a cytochrome P450 enzyme, specifically P450_BM3, which has been engineered and expressed in E. coli BL21 cells. Cytochrome P450 enzymes are heme-containing monooxygenases that play a central role in the metabolism of drugs, xenobiotics, and endogenous substrates. P450_BM3, derived from Bacillus megaterium, is a well-studied, self-sufficient P450 with high catalytic activity, making it an excellent model for enzyme kinetics and biocatalysis studies.

Objective: To measure the absorbance of cell-free extracts containing P450_BM3 using UV-Vis spectrophotometry and to determine the concentration of the heme-bound P450 enzyme using Beer’s Law.

Materials:

UVette Cuvettes (Eppendorf, Cat. #952010051)
Cell-Free Extracts (CFE-A and CFE-B) — provided in tubes labeled “Beer”
Pipettes and Tips
UV/Vis Spectrophotometer (Thermo Scientific, Cat. #840-300000)
Extraction Buffer (50 mM potassium phosphate, pH 7.4) OR 0.25X diluted PBS
- Prepared by diluting from a 1 M stock solution:
- 95 g monobasic potassium phosphate (Carolina, #884250)
- 52.5 g dibasic potassium phosphate (Carolina, #884300)

Procedure:

Thaw the “Beer” tubes containing CFE-A and CFE-B at room temperature.
Pipette 300 µL of CFE-A into a new UVette cuvette. This will be your blank for spectrophotometer calibration.
Pipette 300 µL of CFE-B into another new UVette cuvette.
Measure Absorbance:
- Set the spectrophotometer to 418 nm, the wavelength where heme (bound to P450) absorbs light.
- Use the CFE-A cuvette to blank (calibrate) the machine.
- Measure the absorbance of CFE-B, and record your results.
After measurements, return any remaining sample from the cuvettes back to their original labeled tubes.
Freeze the tubes if you are not proceeding directly to the enzymatic assay.
Rinse UVette cuvettes first with ethanol, then with distilled water, and allow them to air dry.

Post-Lab Questions:

Absorbance Patterns:
Examine the absorbance data you collected.
- Do both CFE-A and CFE-B show a peak at 418 nm?
- Which extract likely contains the P450-expressing cells?
Calculating Protein Concentration (Beer’s Law):
Using Beer’s Law (A = εcL), calculate the concentration of the P450 enzyme:
- Use an extinction coefficient (ε) of 91,000 M⁻¹cm⁻¹ for heme at 418 nm.
- Assume the path length (L) of the UVette is 1 cm.
- Rearrange the formula to solve for c (concentration):
  c = A / (εL)
- Plug in your absorbance values to determine the molar concentration of P450 in your sample.

Learning Objectives

After completing this lab and lecture, you should be able to:

Apply Beer’s Law to calculate protein concentration from spectrophotometric data.
Interpret the UV-Vis absorbance spectrum of cytochrome P450 enzymes.
Determine which samples express active P450 enzyme based on heme absorbance.
Reflect on how buffer composition, enzyme folding, and spectrophotometric setup affect the accuracy of biochemical measurements.

Data Interpretation

Absorbance Peaks:
- At what wavelength did your samples show a peak?
- Which sample(s) displayed a prominent peak at 418 nm?
- Based on the data, which extract likely contains the expressed P450BM3 enzyme?
Enzyme Quantification Using Beer’s Law:
- What is the calculated concentration of your P450_BM3in CFE-A and CFE-B?
  - Use the equation: c=A/εL
    - A = measured absorbance at 418 nm
    - ε = 91,000 M⁻¹cm⁻¹
    - L = 1 cm
Troubleshooting Thought Exercise:
- If one sample had no absorbance at 418 nm, what could be the reasons? Consider protein folding, expression failure, or buffer effects.
- If both samples had similar absorbance, how would you confirm enzyme activity beyond absorbance?
How might mutations in the heme-binding pocket affect the absorbance profile?
Why is it important to use the correct buffer and path length in UV/Vis experiments?
How would you adapt this method to study another heme-containing enzyme?

Learning Objectives

Definition: Term

Beer's Law Spectrophotometric Analysis of P450 Enzyme Expression

Materials:

Procedure:

Post-Lab Questions:

Learning Objectives

Data Interpretation

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