Transcript Density Gradient Centrifugation

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 Provide introductory experience on the theory and
application of centrifugation techniques in the
biological sciences.
 Observe (& work with) a protocol for isolation of plant
mitochondria .
 Provide experience with the theory and application of
marker enzymes.
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Definition:
 A technique (or process) where centrifugal
force is used to separate mixtures of particles
or substances on the basis of their differential
densities.
(Centrifugal Force)
Theory:
 Centrifugal force is applied in a “centrifuge”.
(RPM)
 Angular velocity (RPM) creates centrifugal force.
 centrifugal force is expressed relative to gravity.
 Substances with greater densities will sediment
faster than substances with lesser densities.
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RCF = (1.12 x 10-5) x (r, cm) x RPM2
(r = 10 cm) 
(r = 5 cm)
(r = 1 cm)
(Radius, r)
in cm
Note: RCF (Relative Centrifugal Force) is
expressed as no. of x’s gravity.
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Industrial Decanter
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Armored
Plate
Refrigerant
Vacuum
Drive
Motor
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Biological Material
RNA
DNA
Ribosomes
Nuclei
Chloroplasts
Mitochondria
Endoplasmic reticulum
Lipid bodies
Buoyant
Density (g/cc)
1.90
1.70
1.60
1.32
1.21 – 1.24
1.18 – 1.20
1.11 – 1.12
0.96
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 George Stokes (1851) studied the frictional (drag)
force exerted on spherical bodies as they fell
through viscous liquids.
drag force
 Developed a mathematic expression that relates
all factors that can affect sedimentation velocity.
d2 (
Vs =
 p –  l) g
9
Where:
Vs = settling velocity* of a falling sphere.
d = diameter of the sphere.
p = density of sphere.
l = density of the liquid medium.
 = viscosity of liquid medium.
g = gravitational (centrifugal) force.
gravitational
force
*Vs is also known as “terminal velocity” (Vt)
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 The mathematical ratio of sedimentation
velocity (vt) of a particle to the centrifugal
(gravitational) force causing it to sediment.
 Created by Theodor Svedberg during his
analytical ultracentrifugation studies of
proteins and ribosomes.
 Values of “s” range from 10-13 to 10-11 sec.
 1 Svedberg (S) = 10-13 sec.
s=
vt
g
Where:
vt = terminal velocity of a
falling sphere (cm/sec).
g = gravitational (centrifugal)
force (cm/sec2).
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Particle
Cytochrome c
tRNA
Eukaryotic Ribosomes
Large SU
Small SU
Prokaryotic Ribosomes
Large SU
Small SU
Tobacco Mosaic Virus
Mitochondria
Svedberg Unit
1.7S
4S
80S
60S
40S
70S
50S
30S
200S
20,000-60,000S
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1. Differential Centrifugation
 Cell-free extract and supernatants are centrifuged at
progressively higher speeds and longer times.
2. Density Gradient Centrifugation
 Cell-free extract is centrifuged through a medium whose density
gradually increases toward the bottom of the centrifuge tube.
a) Rate Zonal – sample is centrifuged until most dense
component approaches bottom of centrifuge tube.
b) Isopycnic – sample is centrifuged until all components reach
their equilibrium buoyant density.
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Sample
Low
Density
20% sucrose
(1.08 g/mL)
High
Density
65% sucrose
(1.32 g/mL)
Initial
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• Density of medium <
densities of particles.
• Particles with similar
densities move as
bands towards bottom.
• Particles will/may pellet
out or mix at bottom.
Rate Zonal Centrifugation
• Maximum density of
medium > densities of
any particles.
• Particles move towards
bottom, but stop when
they reach position where
density of medium =
density of particle.
• Sometimes referred to as
equilibrium density
gradient centrifugation.
Isopycnic Centrifugation
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“Shave” off
approximately 15 g
of floret tissue.
Intact Plant
Tissue
• Add 35 ml grinding buffer
• Homogenize thoroughly
• Filter through cheesecloth
Crude
Homogenate
• Centrifuge for 10 min.
@ 500 x g
500g Pellet
(Cell Debris)
500g Suprnt.
(Whole Cell Extract)
• Centrifuge for 30 min.
@ 20,000 x g
20,000g Pellet
(Crude Mitochondria)
20,000g Suprnt.
(Crude Cytosol)
Marker Enzyme Assays
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Definition:
 An enzyme that specifically occurs in only one type of tissue or cell
type, or in only one subcellular compartment (or organelle), and
whose activity can be used as a diagnostic tool to assess certain
aspects related to the tissue, cell or organelle of origin.
Example: Liver / Heart Function Tests.





Alanine transaminase
Aspartate transaminase
Alkaline phosphatase
Gamma-glutamyl transferase
Lactate dehydrogenase
 Creatine kinase
 Troponin
• Activities of one or more enzyme
are measured in blood serum.
• Elevated levels indicate liver
function abnormalities or damage.
• Elevated serum levels indicate
recent heart (attack) damage.
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Succinate dehydrogenase:
 Membrane-bound enzyme of mitochondrion.
 Oxidizes succinate to fumarate in TCA Cycle.
C OO
I
HCH
I
HCH
I
C OO
Succinate
Dehydrogenase
FAD
FADH2
DCPIP
UQH2
Respiratory
Electron
Transport
C OO
I
CH
II
HC
I
C OO
(Blue)
UQ
DCPIPH2
(Colorless)
O2
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Succinate dehydrogenase:
 Membrane-bound enzyme of mitochondrion.
 Oxidizes succinate to fumarate in TCA Cycle.
Phosphoglucoisomerase:
 Soluble glycolytic enzyme of the cytosol.
 Converts glucose-6P to fructose-6P.
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Succinate dehydrogenase:
 Membrane-bound enzyme of mitochondrion.
 Oxidizes succinate to fumarate in TCA Cycle.
Phosphoglucoisomerase:
 Soluble glycolytic enzyme of the cytosol.
 Converts glucose-6P to fructose-6P.
 Can be coupled to glucose-6P dehydrogenase.
↔
PGI:
Fructose-6P
Glucose-6P
G6PDH:
Glucose-6P + NAD+ + H+
↔
6-Phosphogluconate + NADH
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 Measure activities of two marker enzymes in three
cellular fractions (whole extract, mitochondrial fraction,
cytosol fraction).
 Examine the effects of Na-malonate on SDH.
 Make interpretations on purity of fractions.
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