Density gradient centrifugation

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The density is one of the physical separation processes of particles on the basis of sedimentation in a density gradient . Different dissolved macromolecules are sorted in an ultracentrifuge based on their speed of movement ( sedimentation speed ) or density under the influence of strong centrifugal forces .

properties

The speed of movement depends on

Simple gradient mixer for continuous gradients

For density gradient centrifugation, a solvent is required that has a density that increases from top to bottom due to a concentration gradient of a substance dissolved in it (continuous gradient). The concentration gradient runs from bottom to top because the layers of different densities align themselves in the earth's gravitational field and most of the substances used for a gradient have a higher density than the sample. Since the particles to be separated only make up a small part of the aqueous solution in the sample, the density of the sample is slightly higher than the density of water.

The concentration gradient is generated by filling the centrifuge tube with an increasing gradient of a solution. If the order is reversed, the solution mixes as the heavier solution sinks, the gradient sinks to zero. The highest density of the gradient is at the bottom of the centrifuge tube, and each area has a greater density than the one immediately above it (towards the center of the rotor). As a result of the gradient, the migration speed approaches a linear increase with increasing distance from the rotor center; without a density gradient, it increases exponentially with the radius. One possibility of generating a continuous density gradient before centrifugation is the gradient mixer .

In the simplest case of a concentration gradient, the gradient is reduced to only one density level (single-layer discontinuous gradient), the density of which is above the original density of the sample and below the density of the particles to be separated in the sample. By carefully layering solutions with decreasing density, multi-level gradients can be built up; alternatively, solutions that are becoming increasingly dense can be layered under. A gradient mixer is not required to generate discontinuous gradients. The centrifuged particles collect at a suitable density of the gradient at one of the boundary layers between two density ranges.

The sample to be examined is placed on the surface of this solution with the gradient before centrifugation . During the several hours of separation, the molecules sediment in the solution at different speeds. The separation takes place as long as the density of the sample is greater than the density of the solvent and the faster the greater the density difference. After the equilibrium has been established , different bands of the components of the sample are obtained. When a density that corresponds to that of the molecule is reached, the migration ends and the centrifuged sample is in equilibrium. For a precise result, the individual bands must be protected against mixing by convection , which is why they are cooled. Following centrifugation, the bands can mix by diffusion , vibrations and shocks, which is why fractionation is quick and low-vibration.

In density gradient centrifugation, there is an equilibrium of sorting through sedimentation and opposing diffusion for each type of molecule ; smaller molecules have blurred bands. A sedimentation constant K can be determined for each type of molecule . It is defined as the quotient of sedimentation speed and centrifugal acceleration and is given as Svedberg unit (S). So z. B. the bacterial ribosome from two larger subunits, 30 S and 50 S (together 70 S) and that of the eukaryotes from 40 S and 60 S (together 80 S). Smaller viruses such as picornaviruses have a sedimentation constant of 150 S. Viroids are between 5.9 and 8.2 S.

For density gradient centrifugation, rotors with swinging tube holders are better suited than rotors with rigid tube holders. The rotor is allowed to coast down without braking so as not to swirl the density gradient.

Definitions of terms

Fluorescence of DNA with ethidium bromide in a cesium chloride gradient

There are basically two different methods of density gradient centrifugation:

  • In rate-zonal centrifugation , separation takes place in a gradient (synonym zone ) according to the rate of descent (synonym sedimentation rate) or according to the distance covered after a certain time. The centrifugation is stopped before equilibrium is reached. The particles to be separated from each other have a higher density than the densest area of ​​the gradient, therefore a longer centrifugation (to equilibrium) would lead to a precipitate, which would prevent a separation of particles of different but similar densities. The sedimentation rate depends on the size and shape of the particles. Since only the sedimentation rate (and not the density) is important with this method, this centrifugation is called rate-zonal centrifugation . A continuous (steadily increasing) or a discontinuous gradient (with concentration levels) can be used here. An example of this would be ribosomal subunits in a gradient of cane sugar. The bands broaden over time due to diffusion. After a very long time, all of the ribosomal subunits would land on the bottom of the tube as a precipitate. The density of the gradient is chosen so that the bands of the particles to be separated are sufficiently far apart, since an overlap of the bands can lead to wider bands.
  • The separation according to the same density is called isopycnic centrifugation . An example of this would be nucleic acids with ethidium bromide in a gradient of cesium chloride. Only in isopycnic centrifugation do the bands become increasingly sharper over time and remain in the same place on the tube even after a very long period of centrifugation (in equilibrium).

Determination of the molar mass

With a known partial specific volume ν , the molar mass M (in kilograms per mol) can be determined by equilibrium centrifugation . For proteins, this is approximately 0.000735 cubic meters per kilogram, which corresponds to a density of approximately 1360 kilograms per cubic meter.

Alternatively, the molar mass can also be determined using isopycnic centrifugation with the density ρ s at the position of the protein band, the position of the band x s , the density gradient dρ / dx at the position of the protein band (in Kg m −4 ) and the half- width Δx 1 / 2 of the concentration distribution can be determined.

Applications

In biochemistry, density gradient centrifugation is used to separate organelles in a cell in the course of cell fractionation and to determine the molar mass of larger protein complexes . One works here with steep gradients of cane sugar ( sucrose ) or cesium chloride . In cell biology and medicine, rate zonal centrifugation is used to separate PBMC . Synthetic polymers made from sucrose ( Ficoll ) or silica gel ( Percoll ) in isotonic solutions are used as separating solutions for PBMC separation .

literature

  • Paul Reinhart Schimmel, Charles R. Cantor: Biophysical Chemistry: Part II: Techniques for the Study of Biological Structure and Function . HC Freeman Co., San Francisco, 1980, pp. 619-642. ISBN 0-7167-1190-7 .
  • Alfred Pingoud , Claus Urbanke: Working methods of biochemistry . DeGruyter, Berlin 1997, ISBN 3-11-016513-9 ( as a Google book ).

Individual evidence

  1. ↑ N. Guttman, D. Baltimore: Morphogenesis of poliovirus. IV. Existence of particles sedimenting at 150S and having the properties of provirion. In: Journal of virology. Volume 23, Number 2, August 1977, pp. 363-367, PMID 196114 , PMC 515838 (free full text).
  2. ^ A. Hadidi, R. Flores, JW Randles, JS Semancik: Viroids. Csiro Publishing, 2003, ISBN 978-0-643-06789-9 . P. 17.
  3. ↑ MK Brakke: Density gradient centrifugation: A new separation technique. In: J. Am. Chem. Soc. (1951), Vol. 73, pp. 1847-1848.
  4. ^ MK Brakke: Zonal separations by density-gradient centrifugation. In: Archives of biochemistry and biophysics. Volume 45, Number 2, August 1953, pp. 275-290, ISSN  0003-9861 . PMID 13081137 .
  5. ^ PT Sharpe: Methods of Cell Separation , Elsevier, 1998. ISBN 9780080858876 . P. 23f.
  6. ^ Richard Josiah Hinton, Miloslav Dobrota: Density Gradient Centrifugation , Volume 6 of Laboratory Techniques in Biochemistry and Molecular Biology , Elsevier, 1978. ISBN 9780080858753 . P. 13ff.
  7. ↑ JA Miernyk, JJ Thelen: Biochemical approaches for discovering protein-protein interactions. In: Plant Journal (2008), Volume 53 (4), pp. 597-609. doi : 10.1111 / j.1365-313X.2007.03316.x . PMID 18269571 .
  8. ^ JR Patsch, S. Sailer, G. Kostner, F. Sandhofer, A. Holasek, H. Braunsteiner: Separation of the main lipoprotein density classes from human plasma by rate-zonal ultracentrifugation. In: Journal of Lipid Research (1974), Vol. 15 (4), pp. 356-366. PMID 4369164 . PDF .
  9. ^ W. Patsch, JR Patsch, GM Kostner, S. Sailer, H. Braunsteiner: Isolation of subfractions of human very low density lipoproteins by zonal ultracentrifugation. In: Journal of Biological Chemistry (1978), Volume 253 (14), pp. 4911-4915. PMID 209023 . PDF .
  10. ↑ MK Brakke, JM Daly: Density-Gradient Centrifugation: Non-Ideal Sedimentation and the Interaction of Major and Minor Components. In: Science. Volume 148, Number 3668, April 1965, pp. 387-389, ISSN  0036-8075 . doi : 10.1126 / science.148.3668.387 . PMID 17832115 .
  11. ↑ TM Laue: Analytical ultracentrifugation. In: Curr Protoc Protein Sci. (2001), Chapter 7.5. doi : 10.1002 / 0471140864.ps0705s04 . PMID 18429200 .
  12. ^ A b Alfred Pingoud, Claus Urbanke: Working methods of biochemistry , Walter de Gruyter 1997, ISBN 978-3-11-016513-5 , p. 139ff.