Centrifugation and Subcellular Fractionation of Microscopic Components

Centrifugation is a useful means of isolating and purifying cellular components because most organelles and macromolecules differ from one another significantly in size and density (and sometimes also in shape, especially in the case of macromolecules). These properties, as well as the density and viscosity of the surrounding medium, determine the rate at which a specific particle will move when it is subjected to a centrifugal force by being spun at high speed in a centrifuge.

Because of their inherent difference in size, shape, and/or Centrifuge rotor density, the various organelles and other cellular structure can usually be separated, or fractionated, from one another by centrifugation. This process, called subcellular fractionation, enables cell biologists to isolate and purify specific cellular components, which can then be studied in vitro.

For their pioneering work in this area, Albert Claude, George Palade, and Christian de Duve shared a Nobel Prize in 1974. Claude was instrumental in developing differential centrifugation as a way of isolating specific organelles. Palade was quick to use this new technique in studies of the endoplasmic reticulum and the Golgi complex.

This enabled him to establish the roles of these organelles in the synthesis, processing, and secretion of protein. De Duve, in turn, was the discoverer of both lysosomes and peroxisomes. In each case, subcellular fractionation and electron microscopy played vital roles in the detection and characterization of the new class of organelles.

De Duve’s discovery of lysosomes depended on differential centrifugation. His discovery of peproxisomes, depended specifically on equilibrium density centrifugation a powerful technique for resolving organelles and macromolecules from one another based on density differences.

We will discuss each of these techniques, beginning with a consideration of centrifuges and sample preparation in general.

Centrifuges:

In essence, a centrifuge consists of a rotor that is driven by an electric motor. The rotor holds tubes that contain solutions or suspensions of the particles to be separated. Rotors are of two basic types – Fixed-angle rotors have wells in which the tubes are maintained at a specific angle, whereas swinging-bucket rotors have hinges that allows the buckets to swing out as the rotor spins. Many centrifuges have refrigerated chamber to maintain the sample at a specified temperature, usually just above freezing.

Centrifugation at very high speeds (above about 20,000 rpm) requires an ultracentrifuge that has a vacuum system to reduce heating due to air friction and armor plating around the chamber to contain the rotor in the event of an accident, Some ultracentrifuges are capable of speeds up to 80,000 revolutions per minute (rpm), reaching forces up to 500,000 times the force of gravity (g).

Sample Preparation:

Separation of cellular components by centrifugation requires that the tissue first be disrupted, or homogenized, usually in a cold isotonic solution (0.25 M sucrose) is often used for this purpose.

Disruption can be achieved by forcing cells through a narrow orifice, by subjecting the tissue to ultrasonic vibration or osmotic shock, or by grinding in a mortar and pestle, sometimes in the presence of glass in a mortar and pestle, sometimes in the presence of glass beads or other abrasive material.

Depending on the specific organelles to be isolated, a detergent may also be used to solubilize membranes. The resulting homogenate is a suspension of organelles, molecules, and other cellular components. If the tissue has been disrupted gently enough, many of the organelles and other structures in the homogenate are likely to remain intact and retain their original biochemical functions.

Centrifugation Techniques:

Because they differ greatly in size and density, most organelles can be at least partially resolved from one another. The three most common techniques for this purpose are differential centrifugation, density gradient centrifugation, and equilibrium (or buoyant) density centrifugation. The latter two techniques are also applicable for macromolecules.

Differential Centrifugation:

Differential centrifugation is an effective means of subcellular fractionation because organelles differ from one another so much in size and weight that they move, or sediment, at very different rates in response to centrifugal force. As shown schematically, particles that are large and/or dense (purple spheres) sediment rapidly, those that are intermediate in size and/or density (blue spheres) sediment less rapidly, while smaller and/or less dense particles (black dots) sediment still more slowly.

Thus, differential centrifugation takes advantages of differences in sedimentation rate to separate organelles. In fact, one way to express the size of an organelle or molecule is in terms of its sedimentation coefficient, which measures how rapidly the particle sediments when subjected to centrifugation.

Sedimentation coefficients are normally expressed in Svedberg units (S) and are widely used to indicate relative sizes of organelles and macromolecules. (A sedimentation coefficient actually has the units of seconds; the Svedberg unit (S) is defined such that 1 S = 1. 10^-13 sec.)

The tissue of interest is first homogenized, usually in a cold isotonic solution. Subcellular fractions are then isolated by subjecting first the homogenate and then subsequent supernatant fractions to successively higher centrifugal forces and/or to longer centrifugation times.

The supernatant is the clarified homogenate that remains after particles of a given size and density are removed as a pellet by the centrifugation process. In each case, the supernatant from one step is decanted, or poured off, into a new subjected to greater centrifugal force to obtain the next pellet.

In successive steps, the pellets are enriched in unbroken cells and debris; mitochondria, lysosomes, and peroxisomes; ER and other membrane fragments; and free ribosomes and large macromolecules. The material in each pellet can be re-suspended and used for biochemical studies, if desired. The final supernatant contains only soluble cellular components and is called the cytosol.

Each of the fractions obtained in this way is enriched for the respective organelles but is also likely to be contaminated with other organelles and cellular components. Most of the contaminants in a specific pellet can usually be removed by re-suspending the pellet and repeating the centrifugation procedure.

Density Gradient Centrifugation:

Density gradient (or rate-zonal) centrifugation is a variation of the differential centrifugation technique. Instead of being uniformly distributed throughout the solution or suspension at the start, the particles to be separated are present initially as a thin layer on top of a solution containing a solute that increases in concentration, and hence in density, from the top of the bottom of the tube. When subjected to a centrifugal force, particles that differ in size and/or density move downwards as discrete bands, or zones, that migrate at different rates.

The largest and/or densest particles (purple spheres) move into the gradient as a rapidly sedimenting band, while particles that are intermediate in size and/or density (blue spheres) sediment less rapidly, and the smallest particles (black dots) move still more slowly.

Because of the gradient of solute in the tube, the particles at the leading edges of each band continually encounter a slightly more dense Solution and are therefore slowed slightly in their sedimentation. As a result, each band is kept very compact, maximizing the resolution of different-sized particles.

Centrifugation is stopped after the bands of interest have moved sufficiently far into the gradient to be resolved from each other but before any of the bands reaches the bottom of the tube.

Stopping at this point is essential, because the density of the solution is less than the density of any of the particles, even at the very bottom of the tube. If centrifugation were continued too long, the bands would reach the bottom of the tube one after another, pilling up on top of each other and negating the very purpose of the process.

Density gradient centrifugation is widely used for separating both organelles and macromolecules. The tissue of interest is homogenized in cold isotonic solution, and a pellet containing primarily mitochondria and lysosomes is prepared. (For simplicity, we will assume that the tissue contains few peroxisomes.)

The pellet is re-suspended and the suspension is then layered onto a sucrose solution that increases in concentration and density from the top to the bottom of a plastic or celluloid tube. (A glass tube cannot be used because the tube needs to be punctured to collect the bands).

Upon centrifugation, the mitochondria/Top of gradient move into the gradient as a band that sediments more rapidly than the band of lysosomes because mitochondria are both larger and denser than lysosomes. After a suitable length of time, the centrifuge is stopped, the bottom of the tube is punctured, and fractions are collected.

Because the mitochondria move faster than the lysosomes and are therefore closer to the bottom of the tube when it is punctured, a series of mitochondria-enriched fractions will be collected first, followed by a series of lysosome-enriched fractions. By assaying each of the fractions for marker enzymes that are unique to either mitochondria or lysosomes, the fractions containing these organelles can be readily identified and the extent of cross-contamination determined.

Equilibrium Density Centrifugation:

The third major centrifugation technique is called equilibrium (or buoyant) density centrifugation. This procedure also uses a gradient of solute concentration and density, but in this case the solute is sufficiently concentrated such that the density gradient spans the range of densities of the organelles or macromolecules to be separated on it. For organelles, a gradient of sucrose is often used, and the density range is 1.10- 1.30 g/cm³, corresponding to a sucrose concentration range of about 0.75-2.3 M.

For macromolecules, on the other hand, cesium chloride (CsCl) is usually the solute of choice. (For a classic experiment in which CsCl density gradients were used to resolve double-stranded DNA molecules containing the ¹⁴N versus ¹⁵N isotopes of nitrogen (a density difference of about 1%), as well as to detect hybrid [¹⁴N/¹⁵N] DNA molecules at an intermediate density.

The tissue of interest is first homogenized in a cold isotonic solution (0.25 M sucrose), and a pellet containing primarily mitochondria, lysosomes, and peroxisomes is prepared. The pellet is re-suspended is 0.25 M sucrose, and the suspension is layered onto a sucrose gradient that increases in density from 1.10 to 1.30 gm/cm³, a range that include the densities of all three organelles.

Upon centrifugation, the organelles move into the gradient until each reaches its equilibrium, or buoyant, density— the point in the gradient at which the density of the sucrose is exactly equal to the density of the organelle. At its buoyant density, usually represented by r (the Greek letter rho), an organelle has no net force acting on it, so it moves no further.

Given enough time, all organelles will reach their characteristic buoyant density positions in the gradient and will remain there. When the organelles are at their equilibrium positions, the centrifuge is stopped, the bottom of the tube is punctured, and fractions are collected. Because of their differing densities, the three classes of organelles are recovered in different fractions.