Preparative and analytical centrifugation

A brief explanation on Preparative and analytical centrifugation

1. PREPARATIVE AND ANALYTICAL ULTRA CENTRIFUGATION Pratheeba S

2. INTRODUCTION Centrifugation - separation of particles from a solution according to their size, shape, density, viscosity of the medium and rotor speed using centrifugal force. F = m ω 2 r RCF = 1.12 x 10 -5 r.p.m. 2 r Ultracentifugation – very high speed ultracentrifuge is used to precipitate larger biological molecules from solution or separate them by different rate of sedimentation Sediment colloidal solution Separate macro molecules – proteins, nucleic acids Determine the molecular weight Ultracentrifuge – Edward Greydon 50000 – 75000 rpm & yield 1,00,000 – 10,00,000 x g

3. PREPARATIVE ULTRACENTIFUGATION Used to separate subcellular structures & molecules Handle large liquid volumes without any optical readout system 1940s - technical refinement of the preparative centrifugation technique by Claude and colleagues positioned centrifugation technology at the centre of biological and biomedical research for many decades. Preparative centrifuge designed by Pickets in1949. 45000 – 60000 rpm Upto 900000 x g Chamber is sealed, evacuated and refrigerated.

4. Preparative centrifugation is classified based on medium of suspension in which the separation is carried out. Homogenous suspension – Differential centrifugation Suspension medium having density gradients – Density Gradient centrifugation

5. ZONAL CENTRIFUGATION Based on basis of relative velocity Density of the solution increases as we move down the column – different concentration of sucrose Solution should be inert gradient is formed Different bands according to rate of sedimentation Components with high sedimentation rate – lower end of column Shape & size affects sedimentation The different bands are collected by puncturing the tube.

6. APPLICATION Separation of cellular organelles, proteins – antibody. Antibody classes have similar density but different mass, so separation based on density will not resolve the classes RNA-DNA hybrids Ribosomal subunits Virus particles - Bacteriophage LIMITATION The sample occupies a small region of the centrifuge tube at the start of fractionation. The sample becomes less concentrated during fractionation.

7. DIFFERENTIAL CENTRIFUGATION Differential Centrifugation is based upon the differences in the sedimentation rate of biological particles of different size and density. Initially all particles of a homogenate are evenly distributed throughout the centrifuge tube 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. Fragments are divided into different fraction by stepwise increase in applied centrifugal field

8. APPLICATION In bioscience research to separate subcellular organelles (mitochondria, nucleus,ribosomes), and isolation of macromolecules such as DNA,RNA, proteins, or lipids for further studies. In Medicine-for diagnostic purposes Example, purification of influenza virus for studies on fundamental chemical and physical properties of the virus leads to control and prevention of influenza. It can also be used for low-resolution separation of the nucleus. As this technique separates particles based on their sizes, this can be used for the purification of extracts containing larger-sized impurities. LIMITATION Damage, contamination and loss of sample

9. DENSITY GRADIENT CENTRIFUGATION Based on buoyant densities . Independent on velocity , size or shape of particle and time. Sample is loaded to tube with gradient forming solution Particles move to a point where buoyant density = gradient At this point of isodensity no further sedimentation occurs irrespective of time. Also known as Isopycnic centrifugation Simple pelleting of subcellular structures. Gradients used – CsCl, NaI for macromolecules and nucleotides Simple sugars – glucose, sorbitol, glycerol Polysaccharides – dextran, glycogen Protein - BSA

10. APPLICATION Fractionate animal, plant, and bacterial cells, viral particles, lysosomes, membranes, and mac-romolecules in a range of processes. Commercial preparation of viruses for vaccine and immuno-therapy products in both batch and continuous-flow zonal modes.

11. APPLICATION OF PREPARATIVE CENTRIFUGATION

12. Isolation of the microsomal fraction from muscle homogenates and subsequent separation of membrane vesicles with a differing density the isolation of highly purified sarcolemma vesicles outlined and the subfractionation of liver mitochondrial membrane systems

13. ANALYTICAL ULTRACENTRIFUGATION Analytical centrifugation is mainly concerned with the study of purified macromolecules or isolated supramolecular assemblies. the determination of the purity of macromolecules the determination of the relative molecular mass of solutes in their native state the examination of changes in the molecular mass of supramolecular complexes the detection of conformational changes ligand-binding studies

14. PRINCIPLE Process small volume of sample with a built in optical system Molecules are observed opticaly during centrifugation Sample are centrifuged in tubes with quartz windows. As the rotor turns the images of the protein are projected onto a flim or computer. Conc. Of protein is determined by absorbance of light . Result – degree of darkness on flim or deflection of recorder of scanning system Determines the sedimentation coefficient.

15. ANALYTICAL CENTRIFUGE Invented by Svedberg in 1925. Awarded nobel prize in1926. Operates at 60000 – 100000 rpm 50000 – 100000 x g Sample spun is monitored in real time through optical detection system using UV absorption or interference optical refractive index sensitive system Study of hydrodynamic properties of a molecule 3 kinds of optical detection system light absorption system Schlieren system Rayleigh interferometric system

16. ROTOR : Withstand enormous gravitational stress a solid rotor which in its simplest form incorporates one analytical cell and one counterbalancing cell. Counterpoise cell is used to balance the analytical cell. Analytical cell – sector shaped, holds 14mm liquid column, 1m 3 sample upper & lower plane transparent , made of quartz.

17. METHODS OF DETECTION ABSORBANCE (light absorption system) Optima XL-A The instrument possesses increased sensitivity and wide wavelength range; with its high reproducibility, baseline scans may be subtracted to remove the effects of oil droplets on lenses and windows, and of optical imperfections in the windows and lenses. With the absorption optics, the absolute concentration is available in principle at any point. Light source -A high-intensity xenon flash lamp, wavelengths between 190 and 800 nm. The lamp is fired briefly as the selected sector passes the detector . The measured light is normalized for variation in lamp output by sampling a reflected small fraction of the incident light. A slit below the sample moves to allow sampling of different radial positions. To minimize noise, multiple readings at a single position may be collected and averaged. The increased sensitivity of the absorbance optics means that samples may be examined in concentrations too dilute for schlieren or interference optics. E.g, With proteins, measurement below 230 nm allows examination of concentrations 20 times more dilute than can be studied with interference optics

18. SCHLIEREN OPTICAL SYSTEM Light passing through a region in the cell where concentration (and hence refractive index) is changing will be deviated radially, as light passing through a prism is deviated towards the direction normal to the surface. converts the radial deviation of light into a vertical displacement of an image at the camera. Light passing through either pure solvent or a region of uniform concentration will not be deviated radially, and the image will not be vertically displaced in those regions. The schlieren image is thus a measure of the concentration gradient, dc/dr, as a function of radial distance, r. schlieren image Rayleigh interferometric absorbance

19. RAYLEIGH INTERFERENCE OPTICS If the concentration of the reference point, crF, is known, the concentration at any other point can be obtained: Δj - vertical fringe shift a - constant relating concentration to fringe shift.

20. METHODS OF AUC Sedimentation velocity Method High velocity Pellet at the bottom of cell. Sedimentation equilibrium method Low speed Simultaneous sedimentation & diffusion reaches equilibrium Produce gradient.

21. APPLICATION OF ANALYTICAL CENTRIFUGATION to determine the molecular weight of proteins in solution, to examine protein aggregation , to evaluate the molecular shape of proteins to study the interaction of proteins , e.g. between ligands and receptors to obtain insight into biological functions of homologous proteins.

22. Advantages Absolute method. Dispersive method; mixtures are fractionated during analysis. Analytical ultracentrifugation gives access to geometric (size, shape, structure) and thermodynamic properties (equilibrium constants, free energies, enthalpies, entropies). Applicable for a particle size range from 1 to 1000 nm and a wide range of densities. Detection is most versatile due to multiple, synchronous optical systems. Complex mixtures are fractionated with high statistical reliability, as all sedimenting particles are detected. Combination of sedimentation and spectroscopic properties 

23. LIMITATIONS Ultracentrifuges are extremely expensive devices, which require constant maintenance. interacting systems can lead to data that is difficult to interpret if those systems change during the course of the testing.

24. REFERENCE Wilson, PH D. Keith, et al., eds.  Principles and techniques of practical biochemistry . Cambridge University Press, 2000. Ralston, Gregory B.  Introduction to analytical ultracentrifugation . Vol. 1. California:: Beckman, 1993. Nagamani,B “Bioinstrumentation”, Margham Publications, 2016.