Objectives

Introduction

There are two stages of separation with centrifuges. The first stage takes advantage of the difference in the terminal velocities of different particles as determined by Stoke's law:

      vt = 2R2(ps-p)a/(9µ)

In the study of cell organelles, different fractions of the subcellular particles are routinely separated with a centrifuge. It was by this type of isolation technique that mitochondria were discovered to be responsible for the entire tricarboxylic acid cycle and ribosomes accountable for protein biosynthesis.

In a typical separation scheme, an isotonic 0.25-0.35M sucrose solution is mixed with cells. The mixture is placed in a bead homogenizer, an ultrasonic cell disrupter, or a simple kitchen blender and the cell membranes are broken to spill out the cell contents. The resulting mixture of subcellular organelles can be placed in a centrifuge and spun at 1000g for 10 minutes. Intact cells and heavy nuclei are collected at the bottom of the centrifuge tube as a pellet. After the supernatant is further centrifuged at 10,000g for 20 minutes, subcellular particles of intermediate terminal velocities such as mitochondria, lysosomes, and microbodies may be collected. The smaller and lighter particles (ribosomes, endoplasmic reticulum fragments, cell membranes, and microsomes) can be further separated from the supernatant of the preceeding stage by centrifugation at 100,000g for 60 minutes. The final supernatant may be considered to be the soluble portion of the cell cytoplasm. Notice the increasingly longer time and especially the exponentially increasing centrifugation speed required to effect separation. The last speed is achievable in an ultracentrifuge. This mode of separation is commonly called differential centrifugation.

Each fraction obtained through differential centrifugation contains quite a few different types of organelles which have similar sedimentation velocities, i.e. similar values of R²(ps-p). Because this factor is a combination of both the size and the density, the fraction can be further separated based on density alone irrespective of the sizes. This second stage can be accomplished by a process known as density gradient centrifugation.

The density gradient may be established naturally by simply placing sucrose crystals at the bottom of a test tube. The sugar dissolves in the solution and diffuse toward the top. However, the time required to establish a sugar gradient in this manner is unacceptably long, and the process is not well regulated. A more practical method of establishing a density gradient is by placing layer after layer of sucrose solutions of different concentrations, thus, densities, in a test tube, with the heaviest layer at the bottom and the lightest layer at the top. The cell fraction to be separated is placed on top of the layer. A particle will sink if the density of the particle is higher than that of the immediate surrounding solution. It will continue to sink until a position is reached where the density of the surrounding solution is exactly the same as the density of the particle. A centrifuge can be highly helpful to accelerate this process of reaching the quasi-equilibrium point; however, unlike the differential centrifugation technique used during the first stage of cell separation, the length of centrifugation for this second stage does not matter too much, as long as the system is permitted to come to quasi-equilibrium.

    Size and density of some typical organelles*

    ----------------------------------
    Organelle     diameter     Density
                    (µm)       (g/cm3)
    ----------------------------------
    Nuclei          5-10         1.4
    Mitochondria    1-2          1.1
    Ribosomes       0.02         1.6
    Lysosomes       1-2          1.1
    ----------------------------------

    *Carolina Tips, Nov. 1, 1973.

List of Reagents and Instruments

A. Equipment

• Graduated cylinder
• Test tube

B. Reagents

• Sucrose
• Plastic beads of various densities

Procedures

1. Prepare 50 ml of 15% and 50 ml of 40% sugar solutions. Ink or food coloring may be added to each solution to enhance the visual identification of the different layers of sugar solutions.
2. Measure the density of each sugar solution with a hydrometer.
3. Pipet 20 ml of the 15% sugar solution and position the pipet tip at the bottom of the test tube containing 20 ml of water. Carefully let the 15% sugar solution flow out beneath the water layer.
4. Following the above step, pipet 20 ml of the 40% sugar solution and discharge it beneath the 15% solution. There should be three layers: 0%, 15%, and 40%, counting from the top. Thus, one has effectively created a density gradient.
5. Drop a few plastic beads and record the positions where the beads settle.
6. Separate subcellular organelles instead of plastic beads if an ultracentrifuge is available.

Discussions

Questions

1. Based on the level at which the beads float, estimate the density (or the windows of densities) of each type of bead.
2. If 10 cm of 2% sugar solution is placed at the bottom of 10 cm of water in a cylindrical test tube, the equilibrium sugar concentration should be 1%. Find the diffusion coefficient of sugar from literature sources, and calculate the time needed for the surface of the solution to reach 0.95% if the system is left undisturbed.
3. When establishing a density gradient in a test tube, is it better to build from the heaviest layer up or the lightest layer down? Why?
4. Comment on ways to improve the experiment.

References

1. Martin, D. L. and Sampugna, J., Molecules in Living Systems: A Biochemistry Module, Harper and Row, 1973.
2. Mahler, H. R. and Cordes, E. H., Biological Chemistry, Harper and Row, 1966.
3. Howland, J. L., Cell Physiology, Mcmillan, 1973.