Centrifugation PDF

Centrifugation Introduction Centrifugal force, word from Latin centrum, meaning “center”, and fugere, means “to flee”, is the apparent force that draws a rotating body away from the centre of rotation. Biological centrifugation is a process that uses centrifugal force to separate and purify m...

Centrifugation Introduction Centrifugal force, word from Latin centrum, meaning “center”, and fugere, means “to flee”, is the apparent force that draws a rotating body away from the centre of rotation. Biological centrifugation is a process that uses centrifugal force to separate and purify mixtures of biological particles in a liquid medium. It is a method to separate molecules based on their sedimentation rate under centrifugal force. It is a key technique for isolating and analysing cells, subcellular fractions, supramolecular complexes and isolated macromolecules such as proteins or nucleic acids. Particles separate according to their size, shape, density, viscosity of the medium and rotor speed. Types of centrifuge: a. analytical centrifugation : mainly concerned with the study of purified macromolecules or isolated supramolecular assemblies b. preparative centrifugation: devoted to the actual separation of tissues, cells, subcellular structures, membrane vesicles and other particles of biochemical interest. c. Clinical centrifuge d. small-scale laboratory microfuge Principle: Movement of particle under the gravitational force is called sedimentation. When centrifugal force applied by the centrifuge, particles move faster For example, when sand particles added in the water filled bucket it travels slower but it sediment faster when bucket is swung around in a circle.  When a biological sample moves in centrifuge, it experiences an outward centrifugal force. Rate of sedimentation of biological sample is dependent on the applied centrifugal field The applied centrifugal force is determined by the radial distance of the particle from the axis of rotation. 1. Essentially, the rate of sedimentation is dependent upon the applied centrifugal field, G, that is determined by the radial distance, r, of the particle from the axis of rotation (in cm) and the square of the angular velocity, ω, of the rotor (in radians per second) , rotor speed (s) also known as Revolution per minute: G= ω2 r 2. The rate of sedimentation is dependent not only upon the applied centrifugal field, but also on the nature of the particle, i.e. its density and radius, and also the viscosity of the surrounding medium. Stokes’ Law describes these relationships for the sedimentation of a rigid spherical particle: where ν is the sedimentation rate of the sphere, 2/9 is the shape factor constant for a sphere, r is the radius of particle, ρP is the density of particle, ρm is the density of medium, g is the gravitational acceleration and η is the viscosity of the medium. 3. The sedimentation rate or velocity of a biological particle can also be expressed as its sedimentation coefficient (s), whereby: Since the sedimentation rate per unit centrifugal field can be determined at different temperatures and with various media, experimental values of the sedimentation coefficient are corrected to a sedimentation constant theoretically obtainable in water at 20°C, yielding the S20,W value. The sedimentation coefficients of biological macromolecules are relatively small, and are usually expressed as Svedberg units, S. One Svedberg unit equals 10–13 s. What is Relative Centrifugal Force (RCF): Relative centrifugal force is a measurement for the rate of strength in rotors that are of different sizes and types. RCF is the force perpendicular to the surface applied to the sample, which is always in relation to the gravity of the earth. The formula used to calculate the force of centrifugal relative (RCF) could be written as follows: RCF (g Force)= 1.118 × 10-5 × r × (RPM)2 Where r is the radius of the rotor (in centimeters), and RPM is the speed of the rotor in rotation per minute. Although the relative centrifugal force can easily be calculated, centrifugation manuals usually contain a nomograph for the convenient conversion between relative centrifugal force and speed of the centrifuge at different radii of the centrifugation spindle to a point along the centrifuge tube. A nomograph consists of three columns representing the radial distance (in mm), the relative centrifugal field and the rotor speed (in r.p.m.). For the conversion between relative centrifugal force and speed of the centrifuge spindle in r.p.m. at different radii, a straight-edge is aligned through known values in two columns, then the desired figure is read where the straight-edge intersects the third column. When designing a centrifugation protocol, it is important to keep in mind that: the more dense a biological structure is, the faster it sediments in a centrifugal field; the more massive a biological particle is, the faster it moves in a centrifugal field; the denser the biological buffer system is, the slower the particle will move in a centrifugal field; the greater the frictional coefficient is, the slower a particle will move; the greater the centrifugal force is, the faster the particle sediments; the sedimentation rate of a given particle will be zero when the density of the particle and the surrounding medium are equal. Types of rotors Three main types of rotors: 1. fixed-angle rotors 2. vertical tube rotors 3. swinging-bucket rotors Low-speed rotors are usually made of steel or brass, while high-speed rotors consist of aluminium, titanium or fibre-reinforced composites. The exterior of specific rotors might be finished with protective paints. For example, rotors for ultracentrifugation made out of titanium alloy are covered with a polyurethane layer. Aluminium rotors are protected from corrosion by an electrochemically formed tough layer of aluminium oxide.  Fixed-angle rotors Vertical rotors Swinging Bucket Rotor The tubes are held at angle of 14° to 40° They are held vertical parallel to the The rotor swings out to the horizontal to the vertical axis rotor axis. The tubes are held at angle of position when the rotor accelerates. 7° to 10° to the vertical axis Shorter run time. Sedimenting particles have only short The particles move short distance and The particle travels for a longer distance distance to travel before pelleting. the time of separation is shorter than and thus it may allow better separation. fixed angle rotors due to reduced angle Reorientation of the tube occurs during The disadvantage is that the pellet may acceleration and deceleration of the fall back into solution at the end of rotor. centrifugation It is useful for differential centrifugation This kind of rotor can usually hold a they are the method of choice when large number of tubes, resolution of maximum resolution of banding zones is separated bands during isopycnic required such as in rate zonal studies centrifugation is less based on the separation of biological particles as a function of sedimentation coefficient. Types of centrifuges Depending on the particular application, centrifuges differ in their overall design and size. Many different types of centrifuges are commercially available including: 1. large-capacity low-speed preparative centrifuges; 2. refrigerated high-speed preparative centrifuges; 3. analytical ultracentrifuges; 4. preparative ultracentrifuges; 5. large-scale clinical centrifuges; and 6. small-scale laboratory microfuges. Types of centrifugation: On the basis of speed used, It can be divided into three types: Low-speed centrifugation, High-speed centrifugation and Ultracentrifugation. Low-speed centrifugation High-speed centrifugation Ultracentrifugation This centrifugation has a speed in the range of The speed range is 1000-25,000 rpm with The speed of these centrifuges ranges from 1-6000 rpm, with RCF values up to 6000 g 50,000 g. 60,000 to 150,000 rpm. These instruments usually operate at room This is used when higher speeds and Most of the ultracentrifuges are refrigerated temperature with no means of temperature temperature control of the rotor chamber are because this helps in balancing heat produced control of the sample. essential. Rotor chambers are maintained near during intense spinning. 4°C. Two types of rotors, i.e., fixed angle and Three types of rotors are available for high Ultracentrifuges use all three types of rotors, swinging bucket may be used. speed centrifugation-fixed angle, swinging namely, vertical rotors, swinging bucket rotors, bucket, vertical rotors and fixed-angle rotors. The swinging bucket rotor is the most commonly used rotor in ultracentrifuge because this yields the highest concentration of particles. Low-speed centrifuges are especially useful The preparation of biological samples always As compared to high speed centrifuges or for the rapid sedimentation of coarse requires the use of high speed centrifuge. It is microcentrifuges, the ultracentrifuge can precipitates or red blood cells as pellets at the used to sediment cell debris after cell isolate much smaller particles like ribosomes, bottom of the tube homogenization, ammonium sulfate precipitates proteins and viruses. Ultracentrifuges can also of protein, microorganisms and cellular be used to study membrane fractionation. organelles such as chloroplasts, mitochondria and nuclei Depending on the purpose of use, centrifugation can be divided into two types: Preparative Centrifugation is just for a preparative scale separation Analytical centrifugation, analysis of certain parameters are performed. Preparative centrifugation: The preparative-scale separation procedure is simple as it requires the placing of the sample in the tube, inserting the tube in the rotor and spinning the sample for a fixed period. These results in two phases, pellet (the particles which is settled at the bottom of the tube) and the supernatant. Relatively heavy precipitates are sedimented in low speed centrifugation, whereas lighter organelles such as ribosomes require the high centrifugal forces of an ultracentrifuge. It is primarily used for separation and purification of sample for further analysis  Preparative-scale separation makes use of specific method of separation, like the differential centrifugation and density gradient centrifugation. Differential centrifugation Differential centrifugation is based on the variations in the rate of sedimentation of biological particles that vary in dimensions and densities. Consists of successive centrifugation at increasing rotor speeds. Differential centrifugation of a cell homogenate leads to the separation and isolation of the common cell organelles. The large and small particles in the suspension can be separated by centrifugation at different speeds. For example, Initially all particles of a homogenate are evenly distributed throughout the centrifuge tube and then move down the tube at their respective sedimentation rate during centrifugation. The largest class of particles forms a pellet on the bottom of the centrifuge tube, leaving smaller-sized structures within the supernatant. A tissue homogenate which contains the whole cells, nuclei, cytoskeletons, plasma membrane, mitochondria, lysosomes, peroxisomes, microsomes, endoplamic reticulum, small vesicles, large molecules like ribosomes and protein can be separated by differential centrifugation. After a centrifuge run at low speed at 1000 g for 10 min, the heavier particles like nuclei, whole cell, cell debris, plasma membrane will settle down at the bottom of the tube forming pellet. The supernatant thus obtained can be subjected to medium speed centrifugation at 20,000 g for 20 min. Subcellular organelles like mitochondria, lysosomes, peroxisomes will settle as pellet at the bottom of the tube. The supernatant again can be run on a high speed centrifugation at 80,000 g for 1 h, thus settling down miscrosomes and small vesicles as pellet. The supernatant can be further centrifuged to separate out organelles like ribosome from the soluble protein.  Thus differential centrifugation is used to fractionate cell homogenates into their components. The tissue homogenate contains many sub-cellular organelles which differ in size and therefore sediments at different rates. Each pellet is a mixture of different sub-cellular organelles. Therefore, the differential centrifugation is a rough fractionation of the cytoplasmic contents which can be further purified by density gradient centrifugation. Density gradient centrifugation Density gradient centrifugation refers to an approach to separation between molecules in which the separation is determined by the concentration of molecules as they move through a density gradient under the influence of a centrifugal force. Used density gradient solution Density gradient centrifugation relies on the idea that molecules settle under the force of centrifugal forces until they find the same density in a medium similar to their own. The molecules that are denser start to shift towards the bottom of the gradient as they travel through the densities gradient. The molecules are then suspended at a level at which the density of particles is greater than that of the medium. These way molecules of different density are separated into various layers. They can be recovered through various methods. Steps of Density gradient centrifugation: A density gradient in the medium is produced by gently spreading the concentrations with lower levels over more concentrated ones in the centrifuge tube. The sample is placed over the gradient after which the tubes will be then placed inside an ultracentrifuge. The particles move along their gradients until they arrive at a point where their density is equal to with the denseness of medium around them. The fragments are then removed and separated, giving particles that are isolated. The density gradient centrifugation can be divided as a. Zonal centrifugation (separation is depending on size) b. Isopycnic centrifugation (separation is depending on density). Differential centrifugation Density gradient centrifugation Do not use density gradient solution Use density gradient solution Particles settles on the basis of sedimentation rate. Particles settles according to different densities of solution/ media used. Particles were settled when the density of particle and medium is equal. With each phase of rotation, a specific organelle is Organelle is separated in one test tube in different separated in pellet depending on sedimentation layers. rate/rpm. A series of increasing rotation is used in each step. A series of increasing rotation is not used in each step. Easy to use Difficult to use After centrifugation two layers formed – one is pellet, After centrifugation, many layers are formed. and another is supernatant Analytical centrifugation: Relative molecular mass determination For the accurate determination of the molecular mass of solutes in their native state, analytical ultracentrifugation represents an unrivalled technique. The method requires only small sample sizes (20–120 mm3) and low particle concentrations (0.01–1 g dm3) and biological molecules with a wide range of molecular masses can be characterised. At the start of an experiment using the boundary sedimentation method, the biological particles are uniformly distributed throughout the solution in the analytical cell. The application of a centrifugal field then causes a migration of the randomly distributed biomolecules through the solvent radially outwards from the centre of rotation. The movement of the boundary with time is a measure of the rate of sedimentation of the biomolecules. The sedimentation coefficient depends directly on the mass of the biological particle. The concentration distribution is dependent on the buoyant molecular mass. The movement of biomolecules in a centrifugal field can be determined and a plot of the natural logarithm of the solute concentration versus the squared radial distance from the centre of rotation (ln c vs. r2) yields a straight line with a slope proportional to the monomer molecular mass. Alternatively, the relative molecular mass of a biological macromolecule can be determined by the band sedi- mentation technique. In this case, the sample is layered on top of a denser solvent. During centrifugation, the solvent forms its own density gradient and the migration of the particle band is followed in the analytical cell. Molecular mass determination by analytical ultracentrifugation is applicable to values from a few hundred to several millions. It is therefore used for the analysis of small carbohydrates, proteins, nucleic acid macromolecules, viruses and subcellular particles such as mitochondria.  Sedimentation coefficient Conformational changes in biological macromolecules may cause differences in their sedimentation rates, analytical ultracentrifugation represents an ideal experimental tool for the determination of such structural modifications. For example, a macro- molecule that changes its conformation into a more compact structure decreases its frictional resistance in the solvent. In contrast, the frictional resistance increases when a molecular assembly becomes more disorganised. The binding of ligands (such as inhibitors, activators or substrates) or a change in temperature or buffering conditions may induce conformational changes in subunits of biomolecules that in turn can result in major changes in the supramolecular structure of complexes. Such modifications can be determined by distinct differences in the sedimentation velocity of the molecular species. When a new protein species is identified that appears to exist under native conditions in a large complex, several biochemical techniques are available to evaluate the oligomeric status of such a macromolecule. Gel filtration analysis, blot overlay assays, affinity chromatography, differential immuno precipitation and chemical cross- linking are typical examples of such techniques. With respect to centrifugation, sedimentation analysis using a density gradient is an ideal method to support such biochemical data. For the initial determination of the size of a complex, the sedimentation of known marker proteins is compared to the novel protein complex. Biological particles with a different molecular mass, shape or size migrate with different velocities in a centrifugal field. The value of Svedberg units (S 1⁄4 1013 s) lies for many macromolecules of biochemical interest typically between 1 and 20, and for larger biological particles such as ribosomes, microsomes and mitochondria between 80 and several thousand. The prototype of a soluble protein, serum albumin of apparent 66 kDa, has a sedimentation coefficient of 4.5 S.  Figure shows Sedimentation analysis of the dystrophin–glycoprotein complex (DGC) from skeletal muscle fibres. The size of this complex was estimated to be approximately 18S by comparing its migration to that of the standards b-galactosidase (16S) and thyro- globulin (19 S). When the membrane cytoskeletal element dystrophin was first identi- fied, it was shown to bind to a lectin column, although it does not exhibit any carbohydrate chains. This suggested that dystrophin might exist in a complex with surface glycoproteins. Sedimentation analysis confirmed the existence of such a dystrophin–glycoprotein complex and centrifugation following various biochemical modifications of the protein assembly led to a detailed understanding of its compos- ition. Alkaline extraction, acid treatment or incubation with different types of deter- gent causes the differential disintegration of the dystrophin– glycoprotein complex. It is now known that dystrophin is tightly associated with at least 10 different surface proteins that are involved in membrane stabilisation, receptor anchoring and signal transduction processes. The successful characterisation of the dystrophin–glycoprotein complex by sedimentation analysis is an excellent example of how centrifugation methodology can be exploited to gain biochemical knowledge of a newly discovered protein quickly.