Proteomics/Protein Separations - Centrifugation/CENTRIFUGAL FRACTIONATION OF TISSUES AND CELLS: BASIC APPROACHES AND INSTRUMENTATION

1. Some fundamental physical and mathematical concepts
2. Centrifugal fractionation of tissues and cells: Basic approaches and instrumentation
3. Density gradient centrifugation: Preparing gradients and measuring density
4. Density Gradient centrifuation: Rate and isopynic separations of particles

CENTRIFUGAL FRACTIONATION OF TISSUES AND CELLS: BASIC APPROACHES AND INSTRUMENTATION

" separation in excellence" [1]

• Differential Centrifugation

A Technique for Separating organelles or other differently-sized cell components In A centrifuge, Particles of the Same Size and Weight will settle out Into common Layers.

Also, Differential centrifugation is a procedure in which the homogenate is subjected to repeated centrifugations each time increasing the centrifugal force. -Uses equilibrium density gradient: A solution composed of different layers because of the difference in density between the layers. Differential centrifugation separates the homogenate into:

->Nuclei

In cell biology, the nucleus (from Latin nucleus or nuculeus, kernel) is found in all eukaryotic cells and contains the nuclear genes which form most of the cell's genetic material. Nuclei have two primary functions: to control chemical reactions within the cytoplasm and to store information needed for cellular division.

->Mitochondria

In cell biology, a mitochondrion (plural mitochondria) (from Greek mitos thread + khondrion granule) is an organelle, variants of which are found in most eukaryotic cells. Mitochondria are sometimes described as "cellular power plants," because their primary function is to convert organic materials into energy in the form of ATP via the process of oxidative phosphorylation. Usually a cell has hundreds or thousands of mitochondria, which can occupy up to 25% of the cell's cytoplasm. Mitochondria usually have their own DNA, and, according to the generally accepted Endosymbiotic theory, they were originally derived from external organisms.

->Lysosomes

Lysosomes are organelles that contain digestive enzymes (acid hydrolases) to digest macromolecules. They are found in both plant and animal cells, and are built in the Golgi apparatus. At pH 4.8, the interior of the lysosomes is more acidic than the cytosol (pH 7). The lysosome single membrane stabilizes the low pH by pumping in protons (H+) from the cytosol, and also protects the cytosol, and therefore the rest of the cell, from the degradative enzymes within the lysosome. The digestive enzymes need the acidic environment of the lysosome to function correctly. For this reason, should a lysosome's acid hydrolases leak into the cytosol, their potential to damage the cell will be reduced, because they will not be at their optimum pH. All these enzymes are produced in the endoplasmic reticulum, and transported and processed through the Golgi apparatus. The Golgi apparatus produces lysosomes by budding. Each acid hydrolase is then targeted to a lysosome by phosphorylation. The lysosome itself is likely safe from enzymatic action due to having proteins in the inner membrane which has a three-dimensional molecular structure that protects vulnerable bonds from enzymatic attack.

->Microsomes

In cell biology, a microsome is a small vesicle that is derived from fragmented endoplasmic reticulum produced when cells are homogenized. They contain the cell's cytochrome p450 enzymes, involved in oxidative metabolism.

Liver microsomes are used for determination of EROD activity as an indicator of CYP1A1-dependent enzymatic activity. The term microsome refers to a mixture of fragmented endoplasmic reticulum (ER) vesicles present in a cell homogenate after mechanical breakage (homogenization) of tissues such as liver. These microsomes can be concentrated and separated from the other cellular organelles by means of differential centrifugation. Different cellular organelles possess different and unique sedimentation velocities and sediment out of aqueous solution at different centrifugal forces. Unbroken cells, nuclei and mitochondria sediment out at 10,000g, while soluble enzyme and fragmented ER which contains the P450s stay in the solution. At a higher speed, 100,000g, ER sediments out of solution as pellet and the soluble enzymes stay in the supernatant. In this way, P450s in microsomes are concentrated and isolated. Microsomes appear as reddish brown color due to the presence of heme in P450s. P450s are highly abundant in livers of rats, mice and humans.

Technique of getting hepatic cytochrom P-450 from rat:

Rat is sacrificed by cervical dislocation. Liver is excised, chilled, blotted-dry with paper tissue, and weighed. Liver is homoginated using tissue homigenizer with a medium comprising of 0.154 M KCl containing 50 mM tris-HCl (pH 7.4). The homigenate is centrifused at 12000g for 20 minues, supernatant is collected and again centrifused for 1 hour at 105000g. Then, supernatant is discarded and pellet is resuspended in the homogenizing medium and centrifused again for 1 hour at 105000g. Liver microsome obtained from the pellet by resuspendingwith he homogenizing medium, is ready for use for pharmacological studies. All the procedures are performed at 4 degree celcious.

• Basic Centrifuges and Rotors

-->Superspeed and Ultraspeed Centrifuges:

As two different types of preparative centrifuges may be distinguished: superspeed centrifuges and ultraspeed centrifuges or ultracentrifuges.

Superspeed centrifuges generally operate at speeds to about 20,000 rpm, usually do not require evacuation of the rotor chamber, and either drive the rotor directly to through belts or gears.

Ultracentrifuges can be operated at much greater speeds ( upto 65,000 or 75,000 rpm), thus the rotor chamber must be evacuated of air to reduce friction and permit accurate rotor temperature control.

In most ultracentrifuges the rotor is driven either by a motor and a set of gears or by an oil or air turbine system.

-->Swinging-Bucket Rotors:

The most popular and widely used superspeed and ultraspeed centrifuge rotors are the swinging-bucket and fixed-angle rotors.

-->Rotor and Tube Materials:

Early rotors such as the Svedberg rotors were made of steel and ocassionally brass.The high density of these materials and the resulting high rotor weight produces an appreciable load on the centrifuge drive and significantly limits operating speeds.

Most commercial rotors are now made of the partly or entirely of aluminum or titanium.

-->Fixed-Angle Rotors:

Fixed-angle ( or angle head ) rotors are generally simpler in design than are swinging-bucket rotors. In this type of rotor, the centrifuge tubes are held at a specific and constant angle to the horizontal plane that is the tube does not reorient between the vertical and horizontal positions.

• Convection in Swinging-Bucket and Fixed-Angle Rotors

Convection occurs whenever uniform suspensions of particles are sedimented in conventional swinging-bucket and fixed-angle rotors.

The term "convection" means the bulk movement of solute and/or solvent within the centrifuge tube.

The Most common cause of unwanted cinvection is temperature variation in different parts of the centrifuge tube.

• Nature of Tissue Fractions Produced by Differentail centrifugation

The approaches to tissue fractionation taken by Behrens,Bensley, and others in the early 1930s had a major aim the isolation from disrupted cells of one or more identifiable components that could then be physically and chemically characterized.The principal method of analysis was light microscopy. In this sense, their approach was strictly preparative and is to be contrasted with the more analytical procedures of Claude and his successors who were concerned with the purity of centrifugally isolated fractions, examining their enzymatic activities and questioning whether certain properties were unique to specific types of subcellular particles or were more broadly distributed.

• The four major differential fractions.

1. The Nuclear Fraction:

-->The pellet produced by centrifugation of the original homogenate at 600g for 10 minutes.

2. The Mitochondrial Fraction:

-->The pellet obtained when the post-nuclear supernatent (i.e,,the supernatent of the first centrifugation ) is centrifuged at 5000g for 10 minutes.

3. The Microsomal Fraction:

-->The pellet produced when the postmitochondrial supernatent is centrifuged at 50,000g for 60 minutes.

4. The Cytosol or Soluble Phase:

-->The supernatent of the last centrifugation.