# Virtual Laboratory: Subcellular Fractionation

Rationale: To study the function of cellular organelles using the technique of centrifugation.

Background:  Centrifugation and cellular organelles.

We start with the concept of sedimentation.  Sedimentation is the settling out of particles from a liquid suspension.  The settling of a mixture consisting of gravel, sand and water in a beaker is influenced by gravity.

Cell biologists separate cellular organelles.  Subcellular components are much smaller than sand particles by sedimentation due to gravity alone.  An instrument known as a centrifuge is used for this purpose.

A centrifuge is a piece of equipment that accelerates the rate of sedimentation by rapidly spinning the samples, thus creating a centrifugal force many times that of gravity.

Centrifugation is the process of rapidly rotating a receptacle containing a slurry of solid particles in a fluid; the particles are sedimented by a greatly increased gravitational field.

Centrifugal force is the force that pulls a particle away from the center of rotation.  Whenever an object is forced to move in a circular path, centrifugal force is generated.

To appreciate centrifugal force, tie a stone to the end of a string and whirl it rapidly in a circle.  There will be a pull on your hand as the stone rotates.  And, whirl the stone faster, you experience more pull.

Similarly, inside a centrifuge, the faster the sample is spun, the more force is experienced by the sample.  The speed of rotation in a centrifuge is expressed as revolutions per minute (RPM).  The centrifugal force acting on samples in a centrifuge is expressed as relative centrifugal field (RCF) and expressed in units as multiples of the earth’s gravitational field (g).

Differential centrifugation is a mode of centrifugation in which the sample is separated into two phases: a pellet consisting of sedimented material and a supernatant.  Differential centrifugation is based on the differences in sedimentation rate of particles of different size and density.  The tissue homogenate is centrifugally divided into a number of fractions by increasing (stepwise) the applied centrifugal field.  The centrifugal field is chosen so that a particular type of organelle will be sedimented as a pellet, and the supernatant will be centrifuged at a higher centrifugal field for further fractionation.

Differential centrifugation is probably the most commonly used method for the isolation of organelles from homogenized tissue.  However, a limitation of the method is that a pure organelle fraction is theoretically not achievable.  Hence, a quantitative balance sheet is essential for this approach.  Homogeneous organelle fractions may be achieved by density gradient centrifugation, a more involved centrifugation method.

Eukaryotic cells have many compartments, known as organelles, and different cellular functions are carried out in these cellular components.

(Cell structure diagram)

To study these compartments by the technique of subcellular fractionation by differential centrifugation, three steps are involved.  The first one is destructive: to convert the cells into “homogenate”.  The second is to introduce a new order into the system, by grouping together in separate fractions those components of the homogenate with certain physical properties selected by the investigator.   The third step is the analysis of the isolated fractions.

Hypothesis:  Organelles of the cell perform specific functions and hence must have a unique composition of enzymes and proteins.

Virtual Laboratory Procedures: Subcellular fractionation of rat liver organelles.

Rat liver is the most common material used to study subcellular fractionation.

Analysis:

1. Is there a difference between the pattern of acid phosphatase and uricase?
2. Is there a difference between the pattern of acid phosphatase and cathepsin?
3. Is there a difference between the pattern of acid phosphatase and cytochrome oxidase?
4. Is there a difference between the pattern of acid phosphatase and glucose-6-phosphatase?
5. Why is sucrose used in the homogenization medium? How about water?
6. What would be the expected result if a different centrifugation speed has been selected?
7. Why the fractionation steps only enrich for organelles and cannot yield a pure fraction of organelles?

Expected results:
The enzyme distribution pattern shows a novel fraction containing acid hydrolases (some enzyme assays have been performed at acidic pH, pH 5).  This experiment is similar to the one originally described in 1955 for the discovery of the lysosome.  By density gradient centrifugation, the organelle containing uricase can be separated from the lysosome as a separate organelle (peroxisome).  Dr. Christian de Duve, who made this discovery together with his colleagues, was awarded a Nobel prize in 1974 for his discovery of lysosomes and peroxisomes.  Lysosomes serve a wide range of cellular degradative processes and the lack of lysosomal enzymes, such as acid phosphatase, leads to lysosomal storage diseases.

References:
C. DeDuve et al. 1955 Tissue fractionation studies 6. Intracellular distribution patterns of enzymes in rat liver tissue. Biochem J 60, 604.
H.G. Hers & F. van Hoof 1973 Lysosomes and Storage Diseases, Academic Press, New York.
J. Lindsten 1992 Nobel Lectures Physiology or Medicine 1971-1980, World Scientific, Singapore.
J.B. Lloyd & R.W. Mason 1996 Biology of the Lysosome. Plenum, New York.
D.L. Spector et al. 1998 Cells; A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York.
L.A. Seidman & C.J. Moore 2000 Basic Laboratory Methods for Biotechnology, Prentice Hall, New Jersey.

Domain expertise for developing this lab was provided by Dr. Dunne Fong