Purification Protocol

Purification protocol  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.



Purification Step
Vol. (mL) [Protein] (mg/mL) Total Protein (mg)
[Cyt c] (mg/mL) Total Cyt c (mg) Sp. Act.
(Cyt c/
Prot.) Overall Yield
(%)
Tissue Weight ____ - - - - - - 100
Step 2 - Homogenized Sample - - -
Step 7 - Combined Filtrate
Step 11 - Pooled Eluant Fractions
Step 14 - Supernatant
Step 16 - Pooled Eluant Fractions



Protocol

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.

NOTES

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.

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