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 \(\mathrm{B}\) 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 B and C have pI values of \(7.5 .\) Design an experiment to separate enzyme \(A\) from the other two enzymes.

Short answer

Perform differential centrifugation, disrupt mitochondria to separate matrix, use size-exclusion chromatography, and ion-exchange chromatography to isolate enzyme A, confirmed by SDS-PAGE and activity assays.

Step-by-step solution

- Fractionate the cell components

First, isolate the mitochondria and peroxisomes from the cell using differential centrifugation. This involves breaking the cells open (homogenization) and then centrifuging at various speeds to separate cellular components based on their size and density.

- Separate mitochondrial matrix from the membrane

Since enzyme A is in the mitochondrial matrix and enzyme B is in the mitochondrial membrane, disrupt the mitochondria using a hypotonic solution or sonication to release the matrix contents. Then, centrifuge to separate the solubilized matrix (containing enzyme A) from the membrane remnants (containing enzyme B).

- Isolate enzymes by molecular weight

Perform size-exclusion chromatography (gel filtration) to separate enzyme A (60,000 Da), and enzyme C (100,000 Da). The gel matrix will allow smaller molecules (enzyme A) to elute later compared to larger molecules (enzyme C), thus partially purifying enzyme A from enzyme C.

- Use ion-exchange chromatography

Since enzyme A has a pI of 6.5 and enzymes B and C have pI's of 7.5, exploit this difference using ion-exchange chromatography. At a pH below 6.5, enzyme A will carry a net positive charge while enzymes B and C will be less positively charged. Use a cation-exchanger at an appropriate pH around 6.0. Elute enzymes with a salt gradient to separate enzyme A from B and C.

- Validate enzyme purity

Analyze fractions from the ion-exchange chromatography using SDS-PAGE to confirm the presence of enzyme A (based on molecular weight) and absence of enzymes B and C. Further enzymatic activity assays may be carried out to confirm the purity and functionality of enzyme A.

Key concepts

Differential Centrifugation

Differential centrifugation is an essential technique used to separate cellular components based on their size and density. To separate and purify enzyme A, which is located in the mitochondrial matrix, from contaminating enzymes B and C found in the mitochondrial membrane and peroxisome respectively, you first need to fractionate the cell components.
During differential centrifugation, the cells are homogenized to break them open. This mixture is then subjected to a series of centrifugation steps at increasing speeds. Larger and denser organelles like nuclei sediment at lower speeds, while smaller organelles such as mitochondria and peroxisomes require higher speeds to pellet. This method effectively isolates the mitochondria, where enzyme A is found, and the peroxisomes containing enzyme C.

Size-Exclusion Chromatography

After separating enzyme A from enzyme B, size-exclusion chromatography (SEC) can be used to further isolate enzyme A from enzyme C based on their molecular weights. In SEC, also known as gel filtration chromatography, a column is packed with a gel matrix consisting of porous beads.
When a mixture of proteins is applied to the column, smaller proteins (like enzyme A at 60,000 Da) enter the pores of the beads and travel through the column more slowly, eluting later. Larger proteins (such as enzyme C at 100,000 Da) are excluded from these pores and, therefore, pass through the column more quickly. As a result, enzyme A and enzyme C can be separated based on the size difference, leading to partial purification of enzyme A.

Ion-Exchange Chromatography

To exploit the differences in isoelectric points (pI) of enzymes A, B, and C, ion-exchange chromatography (IEC) is highly effective. IEC separates proteins based on their charge at a given pH. Enzyme A has a pI of 6.5, and both enzymes B and C have pI values of 7.5. To separate enzyme A, perform ion-exchange chromatography at a pH below 6.5.
At this pH, enzyme A will carry a net positive charge, while enzymes B and C will be less positively charged or neutral. Using a cation-exchanger column, enzyme A will bind to the negatively charged resin, while enzymes B and C will pass through. A salt gradient can then be used to elute enzyme A from the column, providing further purification.

Mitochondrial Isolation

Mitochondrial isolation is a critical step to obtain enzyme A from the mitochondrial matrix. After breaking open the cells using a homogenizer, differential centrifugation can be used to pellet the mitochondria. The isolated mitochondria still contain two different enzymes: enzyme A in the mitochondrial matrix and enzyme B in the mitochondrial membrane.
To separate these, disrupt the mitochondria in a hypotonic solution or with sonication. This causes the mitochondrial membrane to burst, releasing the contents of the matrix. Subsequent centrifugation sorts the solubilized matrix containing enzyme A from the membrane remnants that house enzyme B.

SDS-PAGE Analysis

Validating the purity of enzyme A after chromatography steps is essential. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is a powerful technique for this purpose. SDS-PAGE separates proteins based on their molecular weight by denaturing them and giving them a uniform negative charge from the SDS.
When the sample is loaded onto a polyacrylamide gel and subjected to an electric field, proteins migrate towards the positive electrode. Smaller proteins move faster and thus further through the gel than larger ones. By comparing the migration of your sample bands to known standards, you can confirm the presence of enzyme A at 60,000 Da and ensure the absence of contaminating enzymes B and C. Additionally, enzymatic activity assays can further confirm the purity and functionality of the isolated enzyme A.