Question

You wish to separate and purify enzyme A from contaminating enzymes \(\mathrm{B}\) and \(\mathrm{C}\). Enzyme \(\mathrm{A}\) is found in the matrix of the mitochondria. Enzyme \(B\) is embedded in the mitochondrial membrane, and enzyme \(C\) is found in the peroxisome. Enzymes A and B have molecular weights of 60,000 Da. Enzyme \(C\) has a molecular weight of 100,000 Da. Enzyme A has a pI of 6.5 Enzymes \(\mathrm{B}\) and \(\mathrm{C}\) have pI values of \(7.5 .\) Design an experiment to separate enzyme A from the other two enzymes.

Short answer

Perform subcellular fractionation, isolate mitochondria, disrupt membranes, centrifuge to remove membrane fragments, and use ion exchange chromatography to separate enzymes based on pI values.

Step-by-step solution

- Subcellular Fractionation

Begin by homogenizing the cell to break it apart and release the cellular components. Use differential centrifugation to separate different organelles. Spin the homogenate at low speed to first remove nuclei and large debris. Then, spin the supernatant at higher speeds to pellet the mitochondria and peroxisomes.

- Isolation of Mitochondrial Matrix

Separate the mitochondrial pellet. Resuspend it in a buffer and use sonication or gentle detergent treatment to disrupt the mitochondrial membranes, releasing enzymes from both the matrix and the membrane.

- Differential Centrifugation for Matrix Extraction

Centrifuge the disrupted mitochondrial mixture at a high speed to remove the membrane fragments. The supernatant will contain enzyme A, while enzyme B will remain with the membrane fragments in the pellet.

- Removal of Enzyme C

Recall that enzyme C is in the peroxisomes. Since peroxisomes pellet separately from mitochondria during differential centrifugation, enzyme C should be absent in the supernatant containing the mitochondrial matrix.

- Gel Filtration Chromatography

Apply the supernatant containing enzyme A and any remaining contaminants to a gel filtration column. Since enzyme A and B have similar molecular weights (60,000 Da), they will not be separated here. Enzyme C (100,000 Da) will elute first, allowing further verification of its absence.

- Ion Exchange Chromatography

Finally, use ion exchange chromatography to separate enzyme A (pI 6.5) from enzyme B (pI 7.5). Choose an anion exchange column if working at a pH above 7.5, or a cation exchange column if working below pH 6.5. Enzyme A will bind or elute based on the column type and the pH used, allowing it to be separated from enzyme B.

Key concepts

Subcellular Fractionation

To begin the process of purifying enzyme A, it's crucial to separate the different cellular compartments where the enzymes are located. This step is known as subcellular fractionation. Start by homogenizing the cells to break them apart and release their contents. Once homogenized, use differential centrifugation. Spin the homogenate at a low speed to remove the nuclei and large debris. Next, spin at a higher speed to separate the mitochondria and peroxisomes, where our enzymes of interest reside. This technique helps in isolating specific organelles, making further separation steps simpler and more effective.

Differential Centrifugation

Differential centrifugation is a technique used to separate organelles and other subcellular particles based on their size and density. After homogenizing the cells, the mixture is subjected to a series of centrifugation steps at increasing speeds:

• Low-speed centrifugation: Removes cell debris and large organelles like nuclei.
• Medium-speed centrifugation: Pellets down mitochondria and peroxisomes.
• High-speed centrifugation: Further separates smaller particles if necessary.

This graded centrifugation allows for stepwise isolation, ensuring that enzyme A from the mitochondrial matrix can be separated from enzyme B embedded in the mitochondrial membrane, and enzyme C from peroxisomes.

Gel Filtration Chromatography

Once enzyme A is isolated in the mitochondrial matrix fraction, we use gel filtration chromatography to remove any remaining contaminants. This method separates molecules based on their size. Since enzyme A and B have similar molecular weights (60,000 Da), they will travel through the gel at similar rates. However, enzyme C, with a molecular weight of 100,000 Da, will elute first.

• Application: Load the supernatant onto the column.
• Elution: Collect fractions; enzyme C will appear first.
• Verification: Confirm the absence of enzyme C in subsequent fractions.

Though enzymes A and B are not separated at this stage due to their similar weights, this step helps verify the absence of enzyme C, streamlining the purification process.

Ion Exchange Chromatography

The final step is ion exchange chromatography, which separates the enzymes based on their isoelectric points (pI). Enzyme A has a pI of 6.5, while enzymes B and C have a pI of 7.5. This difference allows for separation using ion exchange resins:

• Anion Exchange Column: Effective if the buffer pH is above 7.5 because it targets negatively charged proteins.
• Cation Exchange Column: Effective if the buffer pH is below 6.5 because it targets positively charged proteins.

Depending on the chosen pH, enzyme A will bind or elute at different times than enzyme B. By carefully selecting column type and pH, enzyme A can be separated effectively from enzyme B, completing the purification process.