PURIFICATION of CYTOCHROME c

• Prelab
• Purification Protocol
• Results Table
• Notes
• Chromatography
• Column Preparation
• Gel Filtration
• Ion Exchange
• Protein Determination
• Cytochrome Determination
• References
• Return to Biochemistry 353

 Background & Purpose

Cytochrome c, a bright orange pink, iron containing protein which functions as an electron shuttle in the mitochondrial electron transport path, can be purified from beef heart muscle by selective precipitation, ion exchange and/or gel filtration column chromatography.  Cytochrome c is a convenient protein to isolate, because it is quite stable and because its bright color makes it easy to follow during the purification.

The purification protocol that we will use is based on the fact that cytochrome c has several positively charged groups, giving it a pI of around 10. Thus, it is normally bound to the inner membrane of mitochondria by ionic attraction to the negative charges of the phospholipids on the membrane. The tissue and mitochondria are first broken up by homogenization in a blender at low pH, in an aluminum sulfate solution. The positively charged aluminum ions can displace the cytochrome c from the membrane by binding to the negatively charged phospholipids and free the protein in solution. Excess aluminum sulfate is later removed by raising the pH to 8.0, where the aluminum precipitates in the form of aluminum hydroxide.

After filtration to eliminitate the precipitated aluminum hydroxide, we use a ion-exchange chromatography which separates proteins as a function of their charge.  Cytochrome c has several positively charged groups; the column used is made out of  Amberlite CG-50, a negatively charged or cation-exchange resin.

Once the eluant has been collected, ammonium sulfate precipitation is used to selectively precipitate the remaining contaminant proteins in the cytochrome c preparation. This step is based on the fact that most protein precipitate at 80% saturation in ammonium sulfate whereas cytochrome c remains soluble. The excess of salts present in the solution are then removed by gel filtration chromatography which separates protein on the basis of their size.

To assess the success of your purification, samples of the preparation are collected at each step of the purification. These samples are then assayed for total protein content using the Bradford method , and their cytochrome c concentration is measure by spectrophotometry.

 Purification 

Reagents (You have prepared the first three solutions during the first lab session. The other reagents are provided.)

250 mL - 200 mM sodium phosphate, pH 8.0 (10X stock)
1 L - 20 mM sodium phosphate, pH 8.0 (buffer)
100 mL - 20 mM sodium phosphate, 0.5 M NaCl, pH 8.0 (high salt buffer)

50 g bovine heart muscle (or other mitochondria-rich material)
3% Al₂(SO₄)₃ × 18H₂O (w/v)
0.3% Al₂(SO₄)₃× 18H₂O (w/v) (store at 4° C)
2 N NH₄OH
2 N acetic acid

 Pre-lab Preparation

In your notebook, prepare a flowchart of the purification steps described below. Identify the fractions produced at each step and show which fraction contains the cytochrome c. For each step of the purification, remember to measure the volume and collect, as indicated in bold in the procedure, aliquots of solution to determine the appropriate information to fill in the table below.

Week 1:  

1. Separate about 50 grams of muscle tissue and weigh it in a plastic weighing boat.  Suspend the tissue in 120 mL of ice-cold solution of 0.3% Al₂(SO₄)₃ ×18H₂O (w/v).

 2. Using a blender at the highest speed, homogenize the tissue and solution for 1 - 2 minutes. (Note 2).  Measure the volume, and set aside 1 mL to use later to determine the concentration of soluble protein. (See Protein and Cytochrome c  determination for method.)

 3. If necessary, adjust the pH to 4.5 with 3% Al₂ (SO ₄)₃×18H ₂ O (w/v) or with 2 N NH₄OH, and keep the homogenate on ice for 30 minutes, with frequent swirling.

 4. Centrifuge the homogenate for 20 minutes at 8,000 g. Without disturbing the pellet, carefully decant the supernatant from the centrifuge bottle into a 250 mL Erlenmeyer flask. Save both fractions.

 5. Adjust the pH of the supernatant to 8.2 - 8.5 with 2 N NH ₄OH.  Keep at 4° C.

 6. Resuspend the pellet from step 4 in 100 mL of 0.3% Al ₂ (SO₄)₃× 18H ₂ 0 (w/v), and repeat steps 3 - 5, except at room temperature. Combine the supernatant solutions and store at 4° C.

Week 2:

 7. Filter the combined solutions using a coarse filter made from several Kimwipes in a large funnel.  If the filtered solution is turbid, filter again or repeat step 4.  Measure the volume, and save 1mL of the filtrate to determine later the cytochrome c and protein concentration of the solution. (See Protein and Cytochrome c  determination for method.)

 8. Pass the solution through a column (approx. 0.7 cm diameter x 7 cm length) of Amberlite CG-50 cation-exchange resin, prepared and pre-equilibrated with 20 mM sodium phosphate, pH 8.0 buffer as described below (see Column Gel Filtration and Ion Exchange Chromatography and Note 3 for more detail).  The column should contain at least 0.1 mL resin/gram of tissue.  Allow all the solution to flow through the column, but do not let the solution level drop below the top of the column material. Optional: save 1mL of the eluant to verify later that it does not contain cytochrome c. 

 9. Wash the resin with 5 column volume equivalents of 20 mM sodium phosphate, pH 8.0 buffer.

 10. Make sure the top of the column material is flat. Allow the buffer level to drop to just above the top of the column material and stop the column. Carefully add a few mL of the high salt buffer (20 mM sodium phosphate, 0.5 M NaCl, pH 8.0) to the top of the column material, disturbing the column material as little as possible.  Open the valve at the bottom of the column and let the buffer level drop to the top of the column material. Observe carefully what happens in the column. 

 11. Repeat step 10, then add additional high salt buffer and slowly pass 15 mL of high salt buffer through the column, collecting fractions of about 0.5 mL.  Pool the red (cytochrome c) fractions, measure the volume, and save 200 - 500 mL of this solution to determine the cytochrome c and protein concentration of the pooled fractions.

 12. Cool the pooled fractions on ice and add enough solid (NH ₄)₂SO₄ to bring the final concentration to 80% of saturation (see table ). Mix the solution thoroughly to completely dissolve the (NH ₄ )₂SO₄ .  If necessary, adjust the pH to 8.5 (Note 4)

 Week 3:

 13. Centrifuge at 10,000 g for 10 minutes.

 14. Remove and save the red supernatant, discarding the light-colored pellet. Measure the volume, and save 200 - 500 m L of this solution to determine later the cytochrome c and protein concentrations.

 15. Apply the cytochrome c solution to a gel filtration column (Sephadex G-50, see below Column Gel Filtration and Ion Exchange Chromatography for more detail), as described below, and elute with 20 mM sodium phosphate, pH 8.0 buffer. Collect the eluant in approximately 0.5 mL fractions.  Pool the red fractions, measure the volume and determine the cytochrome c and protein concentrations of the solution .

 16. Store the cytochrome c solution at 4 °C, with your name, the date, and sample name written on the container.
 

Note 1: A blender is an easy way to homogenize a sample, but it has drawbacks. Blend too long, and you form a puree with microscopic particulates that will neither filter nor centrifuge. Blend too briefly, and the cells remain unbroken. A homogenizer also heats the solution, so it is wise to cool the solution after every 1-2 minutes of blending.

 Note 2: A column can usually be moved from warm (room temperature) to cold (cold room) without harm, but not moved from cold to warm. The latter causes air pockets to form and these disrupt the buffer flow.

 Note 3: High concentrations of ammonium sulfate may interfere with the Bradford protein assay.

 GEL FILTRATION AND ION EXCHANGE CHROMATOGRAPHY

 Background

 Ion exchange chromatography 

 Gel filtration chromatography 

Gel filtration chromatography separates substances on the basis of their size. "Sephadex," one of the first gel filtration media, is prepared by cross linking dextrans, which are carbohydrate polymers, to produce beads containing an extensive network of channels or pores. If a sample containing a mixture of compounds is applied to a column filled with Sephadex beads, the compounds that are too large to enter the pores are excluded from the interior of the beads and so move down the column rapidly. Conversely, those compounds small enough to enter inside the pores have a much longer potential path through the column and so travel down the column more slowly. The large number of hydroxyl groups renders the gel extremely hydrophilic, and provides a non denaturing environment for most proteins. The G-types of Sephadex differ in their degree of cross linking, and hence in their degree of swelling when hydrated, and in their fractionation range. Sephadex G-50, for example, separates globular proteins in the 1500-30,000 molecular weight range.

Column Preparation

250 mL - 200 mM sodium phosphate, pH 8.0 (10X stock)
1 L - 20 mM sodium phosphate, pH 8.0 (buffer)
100 mL - 20 mM sodium phosphate, 0.5 M NaCl, pH 8.0 (high salt buffer)
~5 mL hydrated volume/column Amberlite CG-50 weakly acidic ion exchange resin (5 mL/g dry wt; equilibrate with 8 vol. 2N NaOH, then buffer until pH ~ 8).

1. Gel chromatography and ion-exchange media have been prepared by the instructor, and you should already have the following 3 solutions stored in the fridge:
 

 2. Obtain the column material from the instructor. Make sure that the column and column material are at room temperature. Temperature gradients will make the column pack and flow unevenly, preventing good separations.

 3. Assemble the columns, and fill about 1/4 full of buffer (see the demonstration column in the lab).

 4. Suspend the column material in excess buffer by swirling, and pour it into the column until the column is completely full.

 5. Open the valve at the bottom of the column.

6. Allow the column material to settle. Add additional column material until the packed bed is the desired height (~10cm). Do not let the column dry out in between each addition.

7. Add buffer, making sure that new buffer (20 mM sodium phosphate, pH 8.0) is added frequently enough to keep clear solution above the top to the column material.

 8. Allow at least two column volumes of buffer to pass through the column. Fill the top part of the column with buffer, cap it and close the valve at the bottom.

Sample Application & Elution

Ion exchange chromatography

For ion exchange chromatography, the sample solution volume can be large, and the solution added simply by using it to replace the column buffer. However the sample solution must not prevent the protein from binding to the column material.

 1. Apply the sample by replacing the column buffer with the sample solution and allowing the solution to flow through the column. This can be done "automatically" by dipping the piece of tubing plugged into the cap of the column directly into the sample bottle. Initiate the flow using a syringe to pull the liquid into the tubing and place the sample solution bottle as high as possible above the level of the column to maintain the flow rate (see demonstration in lab).

 2. After applying the sample, wash the column with several (2 - 5) column volumes of buffer to remove any unbound or weakly bound proteins.

 3. Allow the buffer to drain down exactly to the top of the column material.

 4. Elute the protein by carefully adding the high salt buffer to the top of the column. Add about 1 mL/cm² of column surface. Touch the sample applicator to the wall of the column as you add the buffer to keep from disturbing the top of the column bed.

 5. Allow the high salt buffer to drain into the column, but do not let any column material dry.

6. Add another volume of high salt buffer, then keep the column filled with buffer, being careful to not disturb the top of the column material.

 7. Collect the eluant in several small fractions (~0.5 mL).
 

Gel filtration chromatography

For gel filtration chromatography, it is important to apply the sample to the top of the column evenly, in as small a volume as possible. This will prevent excessive dilution and poor separation.

 1. Allow the buffer to drain down exactly to the top of the column material.

 2. Carefully add about 1 mL of the sample/cm² of column bed to the top of the column. Touch the sample applicator to the wall of the column as you add the sample to keep from disturbing the top of the column bed.

 3. Allow the sample to drain into the column, but do not let any column material dry. Carefully add a volume of buffer equal to the sample volume, using the sample applicator and start collecting .

 4. Still collecting, add another volume of buffer, then fill the column with buffer, being careful to not disturb the top of the column material.

 5. Collect about 30 fractions  (~0.5 mL)

 Sample Collection

   Sample elution can be monitored by color, if it is a colored protein, (hence chromatography) or by its UV absorbance for colorless proteins.  UV-detectors and fraction collectors are frequently useful for slow columns and/or colorless proteins.  By choosing a colored protein and a short, fast column we just avoided these difficulties.

1. Select the fractions that contain cytochrome c on the basis of their color (anywhere from red to pinkish).

2. Pool the fractions for which the color is the most pronounced, and use the least colored ones (from the selection made in step1 ) to rinse all the tubes, in order to maximize the amount of cytochrome c collected.

3. Store the cytochrome c solution frozen at -20°C, with your name, the date, and sample name written on the container.

1. Dickerson, R. & Timkovich, R. 1975 in The Enzymes, 3rd Ed. Vol XI P. Boyer, ed., Academic Press, New York. p 397 - 472.

2.  Margoliash E and Walasek OF. 1967. "Cytochrome c from Vertebrate and  Invertebrate Sources" Methods in Enzymology. 10 : 339-349.
 
3. Nicholls, P. 1974. "Cytochrome c binding to enzymes and membranes" Biochim. Biophys. Acta, 346 261-310.