5: Activity 2-1 - Purification via Ammonium Sulfate Saturation of Cell-Free Extract
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- Explain the principles of protein solubility and how it changes with salt concentration.
- Define and apply the concept of "salting out" using ammonium sulfate.
- Describe the steps involved in partial protein purification from a cell-free extract.
- Calculate the correct amount of ammonium sulfate needed for different saturation levels.
- Understand the importance of temperature control and sample labeling during enzyme purification.
- Cell-Free Extract (CFE): A solution containing cellular components (including proteins) obtained after lysing cells and removing debris.
- Salting Out: A technique to precipitate proteins from a solution by increasing salt concentration, reducing protein solubility.
- Ammonium Sulfate Precipitation: A common method to fractionate proteins based on solubility by gradually increasing salt levels.
- Protein Solubility: The ability of a protein to stay dissolved in solution, influenced by factors like salt, pH, and temperature.
- Precipitate: The solid that forms when proteins aggregate and fall out of solution during salting out.
- Supernatant: The clear liquid remaining above the precipitate after centrifugation.
- Dialysis: A method used to remove small molecules (e.g., salts) from a protein solution using a semi-permeable membrane.
- Centrifugation: A technique that separates components of a mixture based on density by spinning at high speeds.
- Why do proteins become less soluble at high salt concentrations?
- What physical changes would you expect to see as proteins begin to precipitate out?
- Why is it important to keep enzymes like Cytochrome P450BM3 on ice?
- Always label your tubes with enzyme fraction, initials, and date.
- Keep samples cold at all times to prevent denaturation.
- Add ammonium sulfate slowly and gently—foaming = protein damage!
- Record your weights and volumes carefully—this ensures accurate calculations.
- Keep track of which fraction (0–30%, 30–60%, 60–100%) you’re working with.
Activity 2-1: Ammonium Sulfate Saturation of Cell-Free Extract Containing Cytochrome P450BM3
In this activity, you will begin the process of purifying the enzyme Cytochrome P450BM3 from a crude cell-free extract using a method called ammonium sulfate precipitation, or more commonly, "salting out." This is a foundational protein purification technique that takes advantage of how protein solubility changes in the presence of salt. Think of proteins in a solution like people at a party: when the environment is balanced, everyone moves freely and comfortably (soluble). But if the space becomes too crowded or uncomfortable—say, by adding too much salt—it disrupts the atmosphere, and some people (proteins) will leave the party (precipitate out). In technical terms, proteins are more soluble at low or moderate salt concentrations due to the stabilization of charges on their surfaces (this is called "salting in"). However, as salt concentration increases further, the salt starts to "hog" the water molecules, leaving fewer available to keep proteins dissolved. This causes proteins to aggregate and fall out of solution ("salting out").
Each protein has its own unique solubility profile depending on the type and amount of salt, pH, and temperature. By slowly increasing the concentration of ammonium sulfate, a highly soluble salt, you can selectively precipitate different proteins at different stages. This helps separate the target enzyme, Cytochrome P450BM3, from other proteins in the mixture. Ammonium sulfate is especially useful for this kind of purification because:
- It is very soluble in water (up to 4.1 M at 20°C).
- It creates a high ionic strength environment needed for salting out.
- It often stabilizes proteins, which is why purified proteins are commonly stored in ammonium sulfate solutions.
However, a key drawback is that the presence of salt can interfere with enzyme activity assays. High salt concentrations can distort assay results or reduce the activity of some enzymes. Therefore, after salting out, we must remove the excess salt from the protein fraction before any further analysis. To do this, we use a technique called dialysis. Dialysis works similarly to a tea bag in water: small molecules (like salt) can pass through the bag, but larger molecules (like proteins) cannot. We seal the protein solution inside a semi-permeable membrane (dialysis tubing) and submerge it in a salt-free buffer. Over time, the salt diffuses out of the tubing into the surrounding buffer, leaving the proteins behind in a salt-free solution.
⚠️ Important Note: Throughout the purification process, especially when collecting enzyme fractions, always keep your samples on ice. Many enzymes, including P450BM3, are sensitive to temperature and can lose activity if left at room temperature for too long.
Objective: To partially purify the enzyme Cytochrome P450BM3 from a cell-free extract (CFE) by using ammonium sulfate precipitation. This process helps separate proteins based on their solubility.
Materials
- Cell-free extract (CFE) containing cytochrome P450BM3 (from previous lab)
- 1.5 mL centrifuge tubes
- 50 mL conical centrifuge tubes (Corning™ 352070)
- Pipettes and tips
- Extraction buffer: 50 mM potassium phosphate, pH 7.4 OR 0.25X PBS
- (Prepared from 1M stock: 95 g monobasic KH₂PO₄ + 52.5 g dibasic K₂HPO₄ in 1L water)
- Dry ammonium sulfate powder (Flinn Scientific #A0062)
- 50 mL and 1L glass beakers
- Stir bar and magnetic stirrer
- Ice and ice bucket
- Weighing boats and electronic balance
- Centrifuge capable of 5000 rpm
🧊 IMPORTANT! Keep all enzyme samples on ice throughout the procedure to prevent enzyme degradation. Loss of activity can occur if enzymes warm up.
Protocol
Initial Set Up
- Thaw your CFE, and estimate the volume of your sample by eyes.
- Thaw on ice is preferred, but if you are in a rush, you can thaw by hand. However, store on ice afterwards.
- Using a pipette, transfer 50 µL of CFE into a 1.5 mL centrifuge tube.
- Label as: "CFE-SDS" and your initial and date
- Freeze this aliquot.
- Place a clean stir bar in a 50-mL beaker, and then pour in your CFE.
- Place this 50-mL beaker inside a 1L beaker filled with ice to keep it cold.
- Start the magnetic stirrer on low speed.
- Avoid any bubbles as it indicates protein denaturation
You’ll add ammonium sulfate in three steps:
- 0–30% (Mostly small, highly soluble proteins)
- 30–60% (P450 BM3 is commonly enriched here)
- 60–100% (Proteins with very high solubility precipitate)
Each step isolates different protein fractions.
Step 1: 0–30% Saturation
- Calculate the amount of ammonium sulfate needed.
- Use a reference table or formula. Example:
- For 10 mL of CFE, the table says 176 g/L is needed. → Add 1.76 g ammonium sulfate.
- Use a reference table or formula. Example:
- Slowly add ammonium sulfate in small portions while stirring gently. As salt is added, the solution will turn cloudy—this means proteins are precipitating.
- After the final addition, stir for 5 more minutes. Avoid foam (indicates denaturation).
- Transfer the mixture to a 50 mL conical tube.
- Balance tubes and centrifuge your mixture at 5000 rpm for 10 minutes.
- Clean out your 50-mL beaker.
- Slowly transfer only the supernatant back into the clean 50-mL beaker.
- Discard the pellet.
Step 2: 30–60% Saturation and 60–100% Saturation
- Repeat the process and calculate the amount of ammonium sulfate needed to increase from 30% to 60%.
- Example: If the table says 198 g/L → For 10 mL, then add 1.98 g.
- Slowly add ammonium sulfate to your supernatant with gentle stirring.
- Stir for 5 more minutes after the final addition.
- Transfer the mixture to a clean 50-mL conical tube and centrifuge at 5000 rpm for 10 min.
- Discard the remaining supernatant.
- Resuspend the pellet in 1 mL of extraction buffer.
- Label and freeze as: "30–60% - initial, [date]"
After completing this activity, you should be able to:
- Justify the use of ammonium sulfate for partial protein purification.
- Interpret results from each precipitation step and predict where your target protein might be found.
- Troubleshoot common errors in salting out and propose corrective steps.
- Explain how dialysis is used to prepare samples for further enzymatic assays.
- Why do different proteins precipitate at different salt concentrations?
- A student accidentally dumps all the ammonium sulfate at once instead of adding it slowly during the 0–40% step. What could happen to their protein, and how might that affect purification?
- What are some signs that your protein may have denatured during the process? What might have caused this?
- If your enzyme is not found in the 0–40% or 40–60% fractions, what does that suggest about its solubility and the strategy you used?
- You now have three protein fractions—what would be the next logical steps in the purification pipeline (e.g., dialysis, column chromatography)? Why?
- A lab is purifying Protein X, which typically precipitates between 40–60% ammonium sulfate. During the 0–40% step, a student accidentally dumps all the salt in at once instead of adding it slowly. What impact could this have on purification? Hint: Think about whether Protein X might precipitate earlier than expected, or if rapid salt addition could affect protein structure or solubility behavior.