All of the procedures given in Chapter One have in common the use of a microscope. The basic principle for all microscopes is that the cell is composed of smaller physical units, the organelles. Definition of the organelles is possible with microscopy, but the function of individual organelles is often beyond the ability of observations through a microscope. We are able to increase our chemical knowledge of organelle function by isolating organelles into reasonably pure fractions.
A host of fractionation procedures are employed by cell biologists. Each organelle has characteristics (size, shape and density for example) which make it different from other organelles within the same cell. If the cell is broken open in a gentle manner, each of its organelles can be subsequently isolated. The process of breaking open cells is homogenization and the subsequent isolation of organelles is fractionation. Isolating the organelles requires the use of physical chemistry techniques, and those techniques can range from the use of simple sieves, gravity sedimentation or differential precipitation, to ultracentrifugation of fluorescent labeled organelles in computer generated density gradients.
Figure 3.1 Devices used for homogenization
Often, the first step in the preparation of isolated organelles is to obtain a "pure" sample for further analysis. Cells which are not attached to others (such as blood or suspension tissue cultures) can be separated if they have distinct shapes, densities or characteristics which can be marked (such as charge, antigen or enzyme presence). Cells which are part of a more solid tissue (such as liver or kidney) will first need to be separated from all connections with other cells. In some cases this can be performed by simply chelating the environment (removing Ca and/or Mg), but in most instances the cells will need to be enzymatically or mechanically disaggregated. This often results in subtle changes to the cells, and at a minimum will disrupt such cell-cell communications as DESMOSOMES and TIGHT JUNCTIONS.
Homogenization techniques can be divided into those brought about by osmotic alteration of the media which cells are found in, or those which require physical force to disrupt cell structure. The physical means encompass use of mortars and pestles, blenders, compression and/or expansion, or ultrasonification.
Many organelles are easier to separate if the cells are slightly swollen. The inbibition of water into a cell will cause osmotic swelling of the cell and/or organelle, which can often assist in the rupture of the cell and subsequent organelle separation. The use of a hypo-osmotic buffer can be very beneficial, for example, in the isolation of mitochondria and in the isolation of mitotic chromosomes.
Perhaps the most common procedures use Ten Broeck or Dounce homogenizers, both of which are glass mortar and pestle arrangements with manufactured, controlled bore sizes. The addition of a motor driven teflon pestle creates the Potter-Elvijem homogenizer. Ultrasonification is a useful adjunct to this procedure, but is often sufficient by itself.
To obtain pure organelles, the cells must be ruptured, so that the cell membrane is broken, but the organelle to be studied is not. The process of rupturing a cell is known as homogenization of the cell. It also varies from simple mortar/pestle grinding (with the aid of sand or glass beads) for many plant materials, to repeated high velocity compression and expansion in what is known as a "French Press." The French Press is very powerful and can disrupt bacteria and viral particles as well. It is favored for use when molecular dissociation is required, such as in the separation of DNA from the nematode worm C. elegans. Often, cell rupture is aided by rapid freezing (in liquid nitrogen) and subsequent application of mechanical forces.
With all forms of homogenization, the shear force must be carefully controlled. Too little and the organelles will not be separated, too much and even the molecules can be broken.
For molecular separations, mechanical blenders are often used, varying in sophistication from household blenders to high speed blenders with specially designed blades and chambers (e.g. a Virtis Tissue Homogenizer). The mechanical procedures are augmented by various organic solvents (for phase separations) and/or detergents to assist the denaturation and separation of molecules (e.g. DNA from histones). When specific molecules are sought, care must be taken to inhibit powerful degradation enzymes (such as RNase when extracting RNA). This can be accomplished by subjecting the specimen to cold temperature, or by adding specific organic inhibitors (Diethylpyrocarbonate for RNase), or both.
For cellular material which is difficult to shear by the above mentioned techniques (plant cells and bacteria), a device known as a "French Press" is ocassionally used. This device forces a slurry of the cells through an orifice (opening) at very high pressures. The rapid expansion of the pressure from within literally "blows" the cells apart. While this technique is not often required, it is the only way to break open some materials. The units have capacities from 1 to 40 ml and can reach pressures of 20,000-40,000 pounds per square inch (psi).
Ultrasonicators have been used with increasing popularity to separate organelles from cells, particularly from tissue culture cells. Light use of an ultrasonic wave can readily remove cells from a tissue culture substrate (such as the culture flask). It can also be adjusted to merely separate cells, or to break open the plasma membrane and leave the internal organelles intact.
Figure 3.1 presents a few of the various devices used for homogenization.
Figure 3.2 Physical Properties of Biological Materials
Once the cells have been homogenized, the various components must be separated. For some materials (whole blood, cells in suspension), this can be accomplished by the simple use of gravity sedimentation. In this procedure, the samples are allowed to sit, and separation occurs due to the natural differences in size and shape (density) of the cells. Red blood cells are denser than white cells, and thus whole blood separates into an RBC-rich bottom layer, an intermediate "buffy coat" layer of WBC's and an upper plasma portion of settled blood samples (an anti-coagulant is added to prevent coagulation, which would interfer with the separation).
Without question, however, the most widely used technique for fractionating cellular components is the use of centrifugal force. Procedures employing low speed instruments with greater volume capacity and refrigeration are known as "preparative" techniques. Analytical procedures, on the other hand, usually call for high speed with a corresponding lower volume capacity. A centrifuge working at speeds in excess of 20,000 RPM is an ultracentrifuge.
Organelles may be separated in a centrifuge according to a number of basic procedures. They can be part of a moving boundary, a moving zone, a classical sedimentation equilibrium, a preformed gradient isodensity, an equilibrium isodensity or separated at an interface. These are briefly diagrammed in Figure 3.2.
Physical Properties of Biological Materials
Figure 3.3 Methods for centrifugal separations
Before undertaking the centrifugal separation of biological particles, let's discuss the particle behavior in a centrifugal force. Particles in suspension can be separated by either sedimentation velocity, or by sedimentation equilibrium. Sedimentation velocity is also known as zone centrifugation and has the advantage of low speed centrifugation and short times, but yields incomplete separations. Sedimentation equilibrium is also known as isopycnic or density equilibration and requires specimens to be subject to high speeds for prolonged periods of time. It has the advantage of separating particles completely.
Particles in solution will accelerate and attain a terminal velocity when subjected to a centrifugal force. This velocity is determined in part by the size, weight, density and shape of the particle, as well as the viscosity of the medium through which it must travel, and, of course, the centrifugal force generated. The terminal velocity is referred to as the sedimentation velocity of the particle and can bew used to measure the size, weight or density of the particle.
The sedimentation velocity (as terminal velocity) is measurable. The terminal velocity is dependent upon the relative centrifugal force (RCF) applied to the particle and is related to a mathematical factor, the sedimentation coefficient or sedimentation constant. This coefficient is given in Svedberg (S) units, so named for the Swedish pioneer of centrifugation theory and operation, T. Svedberg. The S units are measured as fractions of time, specifically 10 sec. The sedimentation coefficient is determined by dividing the terminal velocity by the centrifugal force field strength. The relationship of sedimentation coefficient to the diameter of a particle is visually presented in Figure 3.2.
While a particle is reaching its terminal velocity, it is also effected by diffusion, usually in a direction opposite to its movement under force. This is a movement by Brownian motion and can be mathematically stated as a constant, the diffusion coefficient (D), which is the spread of the molecule divided by time. It is expressed in units, Ficks, which are 10 cm/sec. As noted in Figure 3.2, there is a direct relationship between the diffusion coefficient of a particle and its diameter. Both, in turn, are related to the sedimentation coefficient of the particle.
Both the sedimentation coefficient and the diffusion coefficient are corrected mathematically to express their values at 20° C, with water as the medium through which they move. They are almost never measured in water nor at this temperature, but formulas exist for the conversion to standard conditions.
What is important is that the S value can be measured and will give an important clue as to the physical structure and size of the particle. In practice, the S value is reasonably easy to determine.
The sedimentation coefficient is given by the formula:
S = 1/r × dr/dt (Equation 3.1)
where = angular velocity of the rotor in radians/sec calculated as 0.10472 x RPM
r = the distance between the particle and the center of rotation (mm)
dr/dt = the rate of movement of the particle (cm/sec)
The value of the sedimentation coefficient can be determined by timing the movement (velocity and distance) of a particle in a medium of known viscosity. The simplest means to do this is to centrifuge for a specific time with a known force, and to calculate the distance moved. Far more expensive, but quicker would be to monitor the movement of the particles while they actually are in the centrifugal field. This monitoring can be accomplished through the use of an "Analytical Ultracentrifuge" equipped with Schlierien Optics. Basically, this procedure takes stroboscopic photos of the advancing molecules within the centrifugal field. It is far too expensive an instrument to have in all but the best equipped molecular laboratories, however.
If a sample contains many different particles with differing densities and sizes, they will begin to separate on the basis of those parameters. The large particles will settle to the bottom of a tube faster than the smaller ones. If the relative centrifugal force is gradually increased, the time for the consequent separation of particles can be decreased.
By varying centrifugation force (speed) and time, while maintaining a continous media density, different sizes of particles can be separated on the basis of their size. Large particles, such as whole cells and nuclei are sedimented at low speeds. Mitochondria and chloroplasts require higher speeds and/or longer times of centrifugation. Ribosomes require even greater forces and longer times. Thus, it is possible to design a protocol which first sediments large organelles, and then by increasing the centrifugation time or speed to sediment smaller particles from the same tube. This protocol is known as differential centrifugation, and the process makes use of both time and speed. Since the procedure sediments large organelles first, they are often contaminated by the smaller organelles which start at the bottom of the centrifuge tube.
At the beginning, the pellet area (bottom of tube) will contain both small and large randomly distributed organelles. As the centrifuge is run, larger particles move down the tube, but smaller ones do not move up; the process is based on sedimentation, not flotation. As the process continues, the larger particles are removed and thus the smaller the particle, the purer the isolated fraction will be at later centrifugation steps. Of course, the smaller organelles are separated as both contaminants of the larger organelles, and as sediments of subsequent centrifugations. Thus, if you wish to maximize the collection of smaller organelles, or minimize the presence of smaller organelles in the large organelle fraction, it is necessary to recentrifuge the larger fractions several times and to collect and pool the resulting smaller units.
It is possible, however, to sediment and float the particles simultaneously. If the particles to be separated have differing densities (gm/ml) they can be separated through a medium that allows particles of one density to "float" and particles of higher density to sink to the bottom. Such media can be layered into the centrifuge tube in step gradients or linear gradients.
In either gradient, the particles are centrifuged until they reach a density equal to the media, thus the name equilibrium density separations. This process has been greatly utilized in the analysis of molecular weights for proteins and nucleic acids, since to a great extent, the density of these molecules can be directly related to their size. Equilibrium density is also used to successfuly isolate membranes and other high lipid-containing organelles.
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