Chapter 3 Centrifugation - Sinica

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Chapter 3 CentrifugationBiochemistry and Molecular Biology (BMB) 3.1 Introduction 3.2 Basic Principle of sedimentation 3.3 Types, care and safety of centrifuges 3.4 Preparative centrifugation 3.5 Analytical centrifugationAnalytical Biochemistry (AB) 3.4.3 UltracentrifugationKoolman, Color Atlas of Biochemistry, 2nd edition1

General Steps in Biochemical Separation2

Separation of Macromolecules Chromatography, precipitation Electrophoresis, ultracentrifugation3

Densities of biological materialMaterialDensity (g/cm3)Microbial cells1.05 - 1.15Mammalian cells1.04 - 1.10Organelles1.10 - 1.60Proteins1.30DNA1.70RNA2.004

Introduction (MBM 3.1)Principles of centrifugationA centrifuge is a device for separating particles from asolution according to their size, shape, density,viscosity of the medium and rotor speedIn a solution, particles whose density is higherthan that of the solvent sink (sediment), andparticles that are lighter than it float to thetop. The greater the difference in density, thefaster they move. If there is no difference indensity (isopyknic conditions), the particlesstay steady. To take advantage of even tinydifferences in density to separate variousparticles in a solution, gravity can be replacedwith the much more powerful “centrifugal force”provided by a centrifuge.5

CentrifugationA centrifuge is used to separate particles or macromolecules:-Cells-Sub-cellular components-Proteins-Nucleic acidsBasis of s density difference between theparticles/macromolecules and the medium in which these aredispersed-Dispersed systems are subjected to artificially inducedgravitational fields6

Type 1– Preparative Centrifugation Collect (isolation) material:cell, subcellular structure, membrane vesicles1. Handle larger liquid volumes (i.e.1 to several thousand litres)2. Range of designs3. Typical rotating speed: 500 - 2000rpmImmunofluorescent imaging ofhuman cells (U2OS) with pan7Cadherin antibody

Type 2– Analytical Ultracentrifugation (AUC) Determine the mass, shape and stoichiometry ratio of noncovalent association of macromolecules (protein-protein,small molecule-protein, quaternary structure)骨骼肌1. Rotates at high speeds e.g.30000 rpm1.肌膜肌膜2. The high speeds used insuch devices generateconsiderable amounts ofheat3. Therefore coolingarrangements are requiredin r/c4a90/new page 50.htm8

3.2 Basic Principle of Sedimentation (AB 3.4.3)Relative centrifugal forceF Mω 2 rM: mass of particler: radius of rotation (cm) (iedistance of particle from axisof rotation)ω :Average angular velocity(radians/sec)2π rev min -1ω 60Rev: revolution per minute(r.p.m.)1 revolution 2π radians 3609

Centrifugal FieldG rω2depends on the radical distance of the particle from therotation axis and the square of the angular velocityG 4π2(rev min ) r-1 2360010

Angular Velocity2π rev min -1ω 60rev: revolution per minute (r.p.m.)11

Because rotors areRelative Centrifugal Force different from variousmanufactures, we usef c Mω 2 r (RCF)RCF ω 2 r g -1 RCF valueRCF to represent thefgMg"No. x g"centrifugation force.2(multiples of earth's gravitational force).2π rmp RCF 60 -1 r g RadiusRadiusRadiusMinAveMaxRCF 1.12 x 10-5 x (rpm)2 x rRPM (x1000)Radiusrpm:revolution per minuter: radius of rotorRCF (x1000)12

Relative centrifugal forceRCF 1.12 x 10-5 x (rpm)2 x rrminrmax13

Interacting Forces in CentrifugationSedimenting force, mpω2r, is opposed by.Fcentrifugemp the mass of equal volume of solvent1. Frictional Resistance againstFfriction Fbuoyancyparticle moving through fluid. f.vf frictional coefficient of particle in the solventv particle velocity2. Flotation Force F ms rω2BALANCE between the sedmenting force and counteracting forceNet force –ms)rω((m)ω 2r -- fvfvM p -M2ps14

Sedimentation Coefficient (s), 沉降係數w 2r(mp-ms) - f v 0Theodor Svedberg (1884-1971),Chemist from Sweden1926 Nobel prize1908. He described a new method(ultracentrifuge) of producing colloid particlesand gave convincing evidence of the validity ofthe theory on the Brownian movements15

S Can be considered“Sedimentation Rate” of a particleunder centrifugation force (dr/dt)/(1/ rω2)m particle massf frictional coefficient of the particle in the solventρ density of solutionv particle velocity S is increased for particle of larger mass(because sedimenting force a m(1-vr) S is increased for particle of larger density (equal volume) S is increased for more compact structures (Shape) ofequal particle mass (frictional coefficient is less) S is increased with rotational speedMild, non-denaturing procedure, useful for proteinpurification, and for intact cells and organelles16

Separation by Sedimentation30 kg10 kg10 kgMaterialIronStoneIronStoneHigher density100 kgSedimentationWeight8Cotton1Iron Mass8 Density10 kg30 kg10 kg Shape1100 kg17

Subcellular FractionationDensities and sedimentation coefficients forbiomolecules, cell organelles, and viruses.Require highdensity mediaHigh concentratedCsCl18

SedimentationSolubleproteinDNARNA19

20

Equation used to calculate NOMOGRAMS (BMB Fig. 3.1)for quickly finding RCF at given speed and rotor lfield (xg)Rotorspeed(r.p.m)21

Types of Centrifuge BMB 3.3.1 Maximum speed of sedimentation Presence /absence of vacuum Temperature control refrigeration) Volume of sample and capacity ofcentrifugation tubes22

Microfuge0.5-1.5 cm3, 10,000 gConcentration of protein samples Large-capacity preparative centrifuge5-250 cm3,3,000-7,000 g23

High-speed refrigerated centrifuge5-250 cm3,100,000 gDifferentiation separation of nucleus,mitochondrial, protein precipitate, largeintact organelle, cellular debris Ultracentrifugation5-250 cm3,600,000 gMicrosomal vesicles, ribosomeHas to reduce excessive rotor temperaturegenerated by frictional resistance sealed chamber, evacuated, cooling24

Centrifuge Rotors Fixed Angle RotorSedimenting particles have onlyshort distance to travel beforepelleting. Shorter run time.The most widely used rotor type.(MBM3.3.2) Swinging Bucket RotorLonger distance of travel may allowbetter separation, such as in densitygradient centrifugation. Easier towithdraw supernatant withoutdisturbing pellet.25

Centrifuge Rotors(MBM3.3.2) Fixed Angle Rotor Vertical Tube Rotor SwingingBucket Rotor26

Centrifuge Its Use and Safety (BMB 3.3.4)On December 16, 1998, milk samples wererunning in a Beckman L2-65B ultracentrifugeusing a large aluminum rotor . The rotor faileddue to excessive mechanical stress27

Mechanical stress Always ensure that loads are evenly balanced before a run. Always observe the manufacturers maximum speed and sampledensity ratings. Always observe speed reductions when running high density solutions,plastic adapters, or stainless steel tubes.Corrosion Many rotors are made from either titanium or aluminum alloy, chosenfor their advantageous mechanical properties. While titanium alloys arequite corrosion-resistant, aluminum alloys are not. When corrosionoccurs, the metal is weakened and less able to bear the stress from thecentrifugal force exerted during operation. The combination of stress andcorrosion causes the rotor to fail more quickly and at lower stresslevels than an uncorroded rotor28

Differential CentrifugationBMB 3.4.1 Based on the differences in thesedimentation rate of the biological particlesof different size, shape and density29

Moving Boundary (differential velocity) Centrifugation1)3)2)1) The entire tube is filled with sample and centrifuged2) Through centrifugation, one obtains a separation of twoparticles but any particle in the mixture may end up in thesupernatant or in the pellet or it may be distributed in bothfractions, depending upon its size, shape, density, andconditions of centrifugation3) Repeat sedimentation at different speed30

Differential Velocity Centrifugation cont. Medium: same density The sedimentation speed is determined mainly on thesize, shape of particle. Application: low resolution separation such aspreparation of nucleus31

32

Density Gradient Centrifugation (BMB 3.4.2) Important technique for purifying proteinsand particularly nucleic acids.Two different types of density gradient centrifugation, fortwo different purposes are: Zonal (or Rate Zonal) Centrifugation(Sucrose density gradient centrifugation) Iso-density (Isopycnic) Centrifugation(Caesium chloride density gradient centrifugation)33

Moving Zone Centrifugation12341. Preparation of gradient sucrose density forcentrifugation mediumDensity1 Density2 Density 3 Density 4 DensityAnalyte2.Sample is applied in a thin zone at the top of thecentrifuge tube on a density gradient34

Moving Zone (differential) Centrifugation –cont.3. Under centrifugal force, the particles will beginsedimenting through the gradient in separate zonesaccording to their size shape and densityInsufficient time--------- Incomplete separationOvertime--------------------co precipitation of all analytes35

Iso-density (Isopyncic) Centrifugation(AB3.4.3)1. Preparation of gradient sucrose density forcentrifugation mediumThe gradient density has to cover the range ofdifferent densities of analytes36

Iso-density (Isopyncic) uilibriumIsopycnic Equal density Molecules separated on equilibrium position, NOT by ratesof sedimentation.After centrifugation, each molecule floats or sinks ( redistribution) to position where density equals density of CsC(or sucrose)l solution. Then no net sedimenting force onmolecules and separation is on basis of different densities ofthe particles.37

Comparison of Two MethodsMoving ZoneCentrifugationCentrifugation:Sample:Lower speed, notcomplete sedimented,stop at proper timeIsopyniccentrifugationCompletely sediment to wherethe density is equilibrated, highspeed, long running timeSedimentation RateSedimentation equilibriumSimilar density,different MWSimilar MW,different densityNucleic acid /cell organelleProtein (similar density,but different in MW)38

Density Gradient Centrifugation39

Subcellular Fractionation (BMB 3.4.4)Skeletal Muscle40

Sarcolemma :It is the surface membrane of the entire fiberT-tubular membranesThey contain extracellular fluid (high in Ca and Na ions)They are continuous tubes of sarcolemmal membrane that run through(transversely) the muscle fiber.Sarcoplasmic reticulum The sarcoplasmic reticlum (SR) is the Castore. It is a diffuse membrane structure that surrounds the sarcomere41

Organelle Separation(Different centrifugationvelocity)(Isopyniccentrifugation)42

Step 1- Cell homogenizationTo obtain pure organelles, the cellsmust be ruptured, so that the cellmembrane is broken, but theorganelle to be studied is not. Theprocess of rupturing a cell is knownas homogenization of the cell andthe subsequent isolation oforganelles is fractionation.43

Four Common MethodsUsing gentlemechanical procedures,called homogenization,the plasma membranesof cells can be rupturedso that the cell contentsare released.44

Ruptured cells producing aliquified cellular homogenate45

Step 2-Cell Fractionation by Centrifugation. Repeated centrifugation at progressively higher speeds willfractionate homogenates of cells into their components. In general, the smaller the subcellular component, thegreater is the centrifugal force required to sediment it.46

Contractile Apparatus of MuscleElectronmicrographs ofindividual myosinprotein moleculesMyosin is a major component of the contractileapparatus of muscle. As shown here, it is composedof two globular head regions linked to a commonrodlike tail.47

Step 3- Density Gradient CentrifugationSarcolemma肌纖維膜48

Step 4- Collection of Fractions Manual collection by pipette Automatic fraction collector for unstablegradient Freezing and slicing49

Affinity Purification of MembraneVesicles (BMB 3.4.5) Cross-contamination ofvesicular membraneprotein Inside-out vesicles,right-side-out vesicle,membrane sheet, leakyvesicles Smaller vesicles aretrapped in largevesiclesIn-side-out (cytoplasmic side out)Right-side-out (apoplastic side out)vesicles50

Lectin Agglutination Method(by Lectin-carbohydrate Interaction)Lectin: protein that interact with carbohydrateThere are many carbohydrates on the surface ofcell51

No carbohydrateInside-out: No carbohydrate52

Lectin Agglutination MethodWGA: Wheat germ agglutininSL: SarcolemmaSN: supernatantNo carbohydrate53

Immunoblot Analysis for Verification ofDifferent Subcellular Fractions54

Analytical UltracentrifugationMBM 3.5.1An analytical ultracentrifuge spins a rotor at an accuratelycontrolled speed and temperature. The concentrationdistribution of the sample is determined at known timesusing absorbance measurements. It can determine:Continuously monitor the sedimentation process Purity of macromole Relative molecular mass of solute (within 5% SD) Change in relative molecular mass of supermolecularcomplexes Conformational change of protein structure Ligand-binding study55

Optical System of an AnalyticalUltracentrifugationback to top(Beckman Optima XL-A):This figure displays a schematicdiagram of the BeckmanOptima XL-A absorbancesystem. A high intensity xenonflask lamp allows the use ofwavelengths between 190 and800nm. The lamp is fired brieflyas a selected sector passes thedetector.56

Sedimentation Velocity MethodSedimentation velocity experimentsare performed at high speed toovercome the effect of diffusion. For asedimentation velocity experiment, aninitially uniform solution is placed in acell and a sufficiently high angularvelocity is applied to cause rapidsedimentation of solute towards thecell bottom. As a result, there is adepletion of solute near the meniscus,causing a characteristic spectrum asshown in the following figure. A sharpboundary occurs between the depletedregion and the sedimenting solute (theplateau)57

Determination of Sedimentation Coefficient (s)4000 s6000 s8000 s10000 sThe velocity of the individual particles in SVexperiments cannot be resolved, but the rate ofmovement of the boundary region can bemeasured. From this, the sedimentation coefficient (s)can be determined. Remember, s depends directly onthe mass of the solute particles and inversely on thefrictional coefficient, which is a measure of size of thehttp://www-bioc.rice.edu/bios576/AU/AU Page.html#ausolute particles.58

Sedimentation Equilibrium Methods Sedimentation equilibriumexperiments have a lower rotorspeed than sedimentationvelocity experiments. Soluteparticles do not pellet at thebottom of the cell, but instead theprocess of diffusion opposes theprocess of sedimentation untilafter a period of time, the twoopposing forces reachequilibrium and the apparentconcentration profile does notchange. At equilibrium, theconcentration of the soluteincreases exponentially towardsthe cell bottom. Each columndisplays a different absorbanceprofile, because theconcentrations of sample arevaried in each.59

Sedimentation Analysis ofSupramolecular Protein ComplexThe binding of ligands may induceconformational changes in subunits ofbiomolecules, which changes thesupramolecular structure of complex.60

Chapter 3 Centrifugation Biochemistry and Molecular Biology (BMB) 3.1 Introduction 3.2 Basic Principle of sedimentation 3.3 Types, care and safety of centrifuges 3.4 Preparative centrifugation 3.5 Analytical centrifugation Analytical Biochemistry (AB) 3.4.3 Ultracentrifugation Koolman, Color Atlas of Biochemistry, 2nd edition

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