Page 209 - Color Atlas of Biochemistry

200 Organelles

Centrifugation B. Density gradient centrifugation
Density gradient centrifugation is used to
A. Principles of centrifugation separate macromolecules that differ only
slightly in size or density. Two techniques
In a solution, particles whose density is higher
than that of the solvent sink (sediment), and are commonly used.
In zonal centrifugation, the sample being
particles that are lighter than it float to the separated (e. g., a cell extract or cells) is placed
top. Thegreater thedifferencein density, the on top of the centrifugation solution as a thin
faster they move. If there is no difference in layer. During centrifugation, the particles
density (isopyknic conditions), the particles
hover. To take advantage of even tiny differ- move through the solution due to their
greater density. The rate of movement basi-
ences in density to separate various particles cally depends on their molecular mass (see A,
in a solution, gravity can be replaced with the formulae). Centrifugation stops before the
much more powerful “centrifugal force” pro- particles reach the bottom of the tube. Dril-
vided by a centrifuge.
Equipment. The acceleration achieved by ling a hole into the centrifugation tube and
centrifugation is expressed as a multiple allowing the contents to drip out makes it
of the earth’s gravitational force (g = possible to collect the different particles in
–2
9.81 m s ). Bench-top centrifuges can reach separate fractions. During centrifugation, the
solution tube is stabilized in the tube by a
acceleration values of up to 15000 g,while
highspeed refrigerated centrifuges can reach density gradient. Thisconsistsof solutions of
carbohydrates or colloidal silica gel, the con-
50000 g and ultracentrifuges, which operate centration of which increases from the sur-
with refrigeration and in a vacuum, can reach face of the tube to the bottom. Density gra-
500000 g. Two types of rotor are available in dients prevent the formation of convection
high-powered centrifuges: fixed angle rotors
and swingout rotors that have movable bucket currents, which would impair the separation
of the particles.
containers. The tubes or buckets used for cen-
centrifugation,
Isopyknic
which
takes
trifugation are made of plastic and have to be much longer, starts with a CsCl solution in
very precisely adjusted to avoid any imbalan- which the sample material (e. g., DNA, RNA,
ces that could lead to accidents.
Theory. The velocity (v) of particle sedi- or viruses) is homogeneously distributed. A
mentation during centrifugation depends on density gradient only forms during centrifu-
gation, as a result of sedimentation and dif-
the angular velocity ω of the rotor, its effective fusion processes. Each particle moves to the
radius (r eff , the distance from the axis of rota- region corresponding to its own buoyant den-
tion), and the particle’s sedimentation prop-
erties. These properties are expressed as the sity. Centrifugation stops once equilibrium
has been reached. The samples are obtained
sedimentation coef•cient S (1 Svedberg,
=10 –13 s). The sedimentation coef cient de- by fractionation, and their concentration is
measured using the appropriate methods.
pends on the mass M of the particle, its shape
(expressed as the coef cient of friction, f), and
its density (expressed as the reciprocal
_
density v,“partialspecific volume”).
At the top right, the diagram shows the
densities and sedimentation coef cients for
biomolecules, cell organelles, and viruses.
Proteins and protein-rich structures have
–3
densities of around 1.3 g cm , while nucleic
–3
acids show densities of up to 2 g cm .Equi-
librium sedimentation of nucleic acids there-
fore requires high-density media—e. g., con-
centrated solutions of cesium chloride (CsCl).
To allow comparison of S values measured in
different media, they are usually corrected to
values for water at 20 °C (“S 20W ”).

Koolman, Color Atlas of Biochemistry, 2nd edition © 2005 Thieme
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