CELL FRACTIONATION

Introduction:

All of the procedures given in laboratory two 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.

HOMOGENIZATION

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. You will use osmotic alteration and compression in this laboratory.

Osmotic alterations

Many organelles are easier to separate if the cells are slightly swollen. The inhibition 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.

Compression/Expansion

For cellular material which is difficult to shear by the above mentioned techniques (plant cells and bacteria), glass beads and a vortex can be used to break open cells. Glass beads are added to the cells and then the cell glass bead mixture is rapidly shaken by a vortex. As the beads shake around in the mixture, they bang and crush the cells between them. This literally "blows" the cells apart.

FRACTIONATION

Centrifugation

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 centrifuges 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 velocity, a preformed gradient isodensity, an equilibrium isodensity or separated at an interface. In this laboratory, you will use the classical sedimentation equilibrium method for cell fractionation.

PHYSICAL PROPERTIES OF BIOLOGICAL MATERIALS

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.

Sedimentation Velocity

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 be used to measure the size, weight or density of the particle.

Sedimentation Coefficient

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 10-3 sec. The sedimentation coefficient is determined by dividing the terminal velocity by the centrifugal force field strength. 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.

Sedimentation equilibrium

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 continuos 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.

For more information on cell fractionation, look at pages 162-165 in your textbook (especially figure 4-35).

Procedure:

1. Yeast cells that are given to you will be already spheroplasted. They have been treated with an enzyme called Zymolase to take off the yeast hard cell wall. This causes them to be very osmotically fragile. You will then break them up with glass beads bashing them apart. Add approximately 5 ml of 10 mM Tris buffer (pH 7.5) to your cells. Add a small amount of glass beads to the tube and vortex 3 times for 30 sec, with 2 minutes on ice between each vortex. Lysis can be checked in your microscope by placing a drop of cell suspension on a slide, cover with at cover slip and view. You should see cell remnants and very few intact yeast cells.

2. When the cells are well broken, we are ready to fractionate. First take three 100ul samples of the broken cells. Place these 100 ul samples into each of three separate small microfuge tubes (label them well for your group). Bring them up to the front of the class for freezer storage and later analysis. Decant the solution into a centrifuge tube and try to keep most of the glass beads out of the new tube. The rest of the sample (4.7 ml) will be centrifuged at 3000 rpm (1000 X g) for 5 minutes to pellet cell fragments, unlysed cells and nuclei.

3. After centrifugation, pour the supernate into a new centrifuge tube (for further centrifugation at 10,000 rpm (9,500 X g) for 10 minutes. While this centrifugation is going, resuspend the pellet in 1 ml of Tris buffer (pH 7.5) and put an about 300 ul into each of three separate small microfuge tubes. Bring them up to the front of the class for freezer storage and later analysis. This is your nuclei sample.

4. After centrifugation, pour the supernate into a new centrifuge tube (for further centrifugation at 12,000 rpm (14,000 X g) for 25 minutes. While this centrifugation is going, resuspend the pellet in 1 ml of Tris buffer (pH 7.5) and put an about 300 ul into each of three separate small microfuge tubes. Bring them up to the front of the class for freezer storage and later analysis. This is your mitochondria sample.

5. Take the supernate (cytosol components) and pipet into three large tubes and bring them up to the front of the class for freezer storage and later analysis. Resuspend the pellet (containing plasma membrane) in 1 ml of Tris buffer (pH 7.5) and put an about 300 ul into each of three separate small microfuge tubes. Bring them up to the front of the class for freezer storage and later analysis.