Analytical centrifugation

Analytical centrifugation is a type of ultracentrifugation.

1. ANALYTICAL CENTRIFUGATION Presented by: Megha Varshini Gowda B R

2. Content Introduction Comparison between preparative and AUC Instrumentation Working principle Types of analysis using AUC Sedimentation velocity method Sedimentation equilibrium method Comparison between SV and SE method Applications References

3. Introduction Theodor Svedberg invented ultracentrifuge in 1923 and was awarded Nobel Prize in 1926 for his research on colloids and proteins using ultracentrifuge. Ultracentrifugation is a achieved through rapid spinning, which imposes high centrifugal forces on suspended particles/molecules and causes separation of such matter on the basis of differences in weight. Ultracentrifugation can be categorized as Preparative and Analytical centrifugation . Picture 1: Theodor Svedberg

4. Figure 2 : Analytical ultracentrifuge instrument. Model- Proteome XL-1 The sample can be monitored in real time through a optical detection system. It can run at about 120,000-150,000 rpm and RCF as high as 625,000g. Rotor chambers are evacuated to reduce friction. Equipped with self balance system and microprocessor control. Analytical centrifugation

5. AUC technology can provide more answers questions such as Shape: How spherical is my protein? Diameter: What’s the size of my particle? Mass: What’s the molecular weight of my protein or complex in solution? Stoichiometry : How many subunits comprise my protein? Purity: Are there other particles in my sample? Heterogeneity: Is my protein bound to other molecules, and what’s the configuration of the complex? Association: Does my protein associate and/or dissociate with other proteins? Conformation: Does the conformation of my protein change upon binding to a ligand ? Advantages of AUC

6. Comparison between preparative and analytical ultracentrifugation Preparative ultracentrifugation Large sample size can be used. No optical read-out. collect fractions and analyze them after the run. Less pure sample can be used. Used to estimate sedimentation coefficient and MW.Generally used to separate organelles and molecules. Analytical ultracentrifugation Uses small sample size (less than 1ml). Built in optical system to analyze progress of molecules during centrifugation. Uses relatively pure samples. Used to precisely determine sedimentation coefficient and MW of molecules.

7. Instrumentation and working The main parts of an analytical centrifugation are:

8. Rotors and cells The rotor is solid with holes to hold the sample cells. Each cell contains a centerpiece with channels to hold liquid samples. The centerpiece in turn is sealed between windows to permit the passage of light. Centerpieces are available that hold either 4 or 8 cells. Different analytical cells are available with volume capacities between 0.02 to 1.0 cm 3 . Fig.3: Analytical centrifuge rotor and sample double sector- shaped cell. Fig.4: Rotor

9. Optical system and Data acquisition As the rotor spins, each cell passes through the optical paths of detectors capable of measuring the concentration of molecules at closely spaced radial intervals in the cell. There are three commercially available optical detectors to measure the concentration distributions: Absorbance spectrophotometer Rayleigh interferometer Fluorescence detector. Absorbance spectrophotometer: usable over wavelength range 190-800nm. Under conditions where Beer-Lambert law holds, the absorbance signal is directly proportional to the solute concentration.

10. Fig.6: Schematic diagram of the optical system of an analytical ultracentrifuge.

11. Rayleigh interferometer: signal consists of equally spaced horizontal fringes and does not rely on chromophore , hence colorless compounds (Polysaccharides and lipids) can be characterized. Fluorescence detector: laser light and suitable labels are used in this. A detector collects light absorption data, which a computer digitizes and records. Data obtained will be interpreted using softwares such as ULTRASCAN, SEDANAL, HETEROANALYSIS, SEDFIT/SEDPHAT, SEDNTERP and result is given out.

12. Principle Basic principle remains the same as that of the preparative ultracentrifugation. Mass will redistribute in a gravitational field until the gravitational potential energy exactly balances the chemical potential energy at each radial position. If we monitor the rate at which boundaries of molecules move during this redistribution, then it is sedimentation velocity experiment. If we determine the concentration distribution after equilibrium is reached, then it is a sedimentation equilibrium experiment. Fig.7: Different forces acting on particle.

13. Factors affecting sedimentation More density - faster sedimentation Denser biological buffer system – slower movement of the particle More frictional coefficient - slower movement More centrifugal force- faster sedimentation Sedimentation rate=0. If, density of particle =density of the surrounding medium. Sedimentation coefficient : the velocity of a particle per unit centrifugal field. Where, s = sedimentation coefficient v = velocity of a particle ω = angular velocity of rotation r = distance of a particle to the rotor axis v ω 2 r s =

14. Types of experiments in AUC The two most common types of analysis performed with analytical ultracentrifuges are: Sedimentation velocity experiments. Sedimentation equilibrium experiments.

15. Sedimentation velocity experiments are performed at high speed to overcome the effect of diffusion. An initially uniform solution is placed in a cell and a sufficiently high angular velocity is applied to cause rapid sedimentation of solute towards the cell bottom. As a result there is a depletion of solute near the meniscus, causing a characteristic spectrum . It is performed using 2 sector cells which requires 420 μ L/sample. The ultracentrifuge, detector and computer record the time course of the sedimentation process, yielding information about the shape, mass, and size of the molecules. Sedimentation velocity method

16. Determination of molecular weight: M= RTs D(1-ʋ ρ ) Where, M: anhydrous molecular weight of the macromolecules R: Gas constant T: Absolute temperature s: sedimentation coefficient of the molecule D: Diffusion coefficient of the molecule ʋ: partial specific volume of the molecule ρ : density of the medium Fig.8: Graph obtained after SV experiment.

17. Sedimentation equilibrium method Sedimentation equilibrium experiments involve studying the steady-state equilibrium of the sample in solution. And requires lower rotor speed than sedimentation velocity experiments. Solute particles do not pellet at the bottom of the cell, but instead the process of diffusion opposes the process of sedimentation until after a period of time, the two opposing forces reach equilibrium and the apparent concentration profile does not change. SE are performed in 6 sector centerpieces which require 110 μ L/channel. Sedimentation equilibrium provides the same type of information about the solution molar masses, stoichiometries , association constants and solution nonideality .

18. Fig.9: Concentration profile in sedimentation-diffusion equilibrium. Determination of molecular weight: M= 2RT ln (C 2 /C 1 ) ω 2 (1-ʋ ρ ) (r 1 2 -r 2 2 ) Where, R: Gas constant ω : angular velocity T: Absolute temperature ʋ: partial specific volume of the molecule ρ : density of the solvent C 2 and C 1 : concentration of solute at distances at r 2 and r 1

19. Comparison between the two types of experiment in AUC Sedimentation velocity Sedimentation equilibrium Angular velocity Large (chosen according to the particles’ sedimentation properties) Small Sample required 420 μ L 110 μ L/channel Analysis As a function of time (3-6 hours) At equilibrium (after 24 hours) Measurement Forming a boundary (sedimentation profile as function of time) The particle distribution in the cell Calculated parameters Shape, mass composition Mass composition Graph obtained

20. Applications With Analytical Ultracentifugation (AUC) the following characteristics of a molecule can be determined: Native Molecular Mass Stoichiometry Assiociation Assembly Models Conformation & Shape Diffusion & Sedimentation

21. Native Molecular Mass AUC is the only technique with which you can determine accurately the native molecular weight of a sample. AUC is applicable over a wide range of molecular weights from approx. 2.5 kDa up to 1.5 MDa . A typical experiment takes about 16 hours and should be done overnight. Stoichiometry The stoichiometry of a molecule can be calculated from the determined molecular mass. with high quality data it can be easily established whether the native conformation of a protein is a hexa - or a heptamer . Association The sedimentation equilibrium method is particularly sensitive for the study of relatively weak associations with associations constants (K) in the order of 10-100 M.

22. Assembly Models The assembly of a protein complex can be calculted from the determined molecular mass. Ligand binding can also be analyzed using sedimentation velocity methods if the ligand and acceptor differ greatly in their sedimentation coefficients. Alternatively, a thermodynamic analysis may be made using sedimentation equilibrium methods. Conformation & Shape Information about the shape and the conformation of a protein as well as the interaction between macromolecules can be obtained from the sedimentation and diffusion coefficients obtained from a sedimentation velocity experiment. Sedimentation coefficients are particular useful for monitoring changes in conformation of a protein.

23. References Wilson, K., Walker, J. Principles and Techniques of Practical Biochemistry .7 th edition 2010. Cambridge university press, New York. Pp: 95-99. Upadhyay , A., Upadhyay , K. Biophysical chemistry . Himalaya publishing house, Delhi. Pp: 321-330. Serdyuk , I.N., Zaccai , N.R. Methods in Molecular Biophysics . 2007. Cambridge university press, UK. Pp: 339-385. Talluri , S. Bioanalytical Techniques . IK ineternational publishing house Pvt.Ltd , New Delhi. Pp:22-28. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2711687/#!po=75.7813