Mitochondria generate the vast majority of the chemical energy that sustains life. This function is accomplished through a highly organized internal structure. To understand how this organelle works, scientists must separate it from the rest of the cell and then break it down into its constituent parts, a methodical process known as mitochondria fractionation. This technique allows researchers to isolate and study the distinct membranes and internal fluid, revealing the precise location and function of the thousands of molecules that drive cellular metabolism. Fractionation is a foundational technique in cell biology, providing the physical samples necessary to map metabolic pathways and investigate the origins of mitochondrial disease.
The Complex Structure of Mitochondria
Fractionation is necessary because the mitochondrion is a highly structured organelle defined by four distinct compartments, each hosting specialized reactions. The outer membrane acts as a protective barrier, containing channel-forming proteins called porins that permit the passage of small molecules. The intermembrane space is the narrow region between the two membranes, serving as a temporary reservoir for protons pumped from the organelle’s interior.
The inner membrane is highly convoluted, folding inward to form cristae, which significantly increase the surface area for chemical reactions. This membrane is the site of oxidative phosphorylation, the process that generates adenosine triphosphate (ATP), and contains the protein complexes of the electron transport chain. Enclosed by the inner membrane is the matrix, a dense fluid containing mitochondrial DNA, ribosomes, and hundreds of enzymes. The matrix is where the Krebs cycle takes place, supplying the carriers needed for the inner membrane’s energy production machinery.
Separating Mitochondria from the Cell
The first stage of fractionation involves isolating intact mitochondria from the surrounding cellular material, beginning with cell disruption. Cells are broken open through homogenization, where tissue is ground or cells are rapidly sheared in a specialized device like a Dounce homogenizer. This mechanical force ruptures the outer cell membrane, releasing the organelles and cellular fluid (cytosol). The resulting mixture, or homogenate, contains nuclei, cell debris, and all the organelles.
Separation relies on differential centrifugation, which separates components based on their size and sedimentation rate. The homogenate is first subjected to a low-speed spin (around 600 to 1,000 times the force of gravity) to pellet the largest components, such as unbroken cells and nuclei. The remaining liquid, the supernatant, is carefully removed and centrifuged again at a higher speed (up to 10,000 times the force of gravity). This forceful spin causes the mitochondria to form a pellet at the bottom of the tube, separating them from lighter components like the endoplasmic reticulum and cytosol that remain in the liquid.
Isolating Sub-Mitochondrial Components
Once intact mitochondria are obtained, the second stage of fractionation separates the organelle into its sub-components. This begins by selectively breaching the outer membrane using a mild detergent, such as digitonin, or osmotic shock. The outer membrane is more susceptible to disruption than the inner membrane. This controlled disruption strips away the outer membrane and releases the contents of the intermembrane space, leaving behind a structure known as a mitoplast—the inner membrane enclosing the matrix.
A high-speed spin separates the components: soluble outer membrane fragments and intermembrane space contents remain in the supernatant, while the heavier mitoplasts form a pellet. To separate the inner membrane from the matrix, the mitoplasts are forcefully disrupted, often through sonication, which uses high-frequency sound waves. The resulting mixture is then subjected to ultracentrifugation at extremely high forces, sometimes exceeding 100,000 times the force of gravity. This intense spinning causes the insoluble inner membrane to form a pellet, while the soluble enzymes and DNA of the matrix remain in the final supernatant, yielding purified fractions for detailed biochemical study.
Scientific Discoveries Aided by Fractionation
Mitochondria fractionation has been foundational in mapping the complex metabolic architecture of the cell. Early applications pinpointed the location of specific enzymes, confirming that the electron transport chain components are physically embedded in the inner membrane. By analyzing the protein content of purified fractions, researchers assigned the enzymes of the Krebs cycle to the matrix and those involved in lipid synthesis to the outer membrane. This localized understanding of function provided the framework for modern biochemistry.
Fractionation remains a powerful tool for investigating cellular dysfunction in the context of disease. Scientists use this technique to analyze how the proteome—the entire set of proteins—of each mitochondrial compartment changes in conditions like cancer, neurodegenerative disorders, and metabolic syndromes. Isolating a specific fraction allows for the study of how drug candidates affect a single component. For instance, researchers can test a compound’s influence on the ATP-generating enzymes in the inner membrane fraction, providing high-resolution insights into the organelle’s role in health and disease.