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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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 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 is applied in a “centrifuge”.


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.
(Centrifugal Force)
(RPM)
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(Radius, r) in cm
RCF = (1.12 x 10 -5 ) x (r, cm) x RPM 2
(r = 10 cm)

(r = 5 cm)
(r = 1 cm)
Note: RCF (Relative Centrifugal Force) is expressed as no. of x’s gravity.
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Industrial Decanter
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Refrigerant
Drive
Motor
Vacuum
Armored
Plate
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 George Stokes (1851) studied the frictional (drag) force exerted on spherical bodies as they fell through viscous liquids.
 Developed a mathematic expression that relates all factors that can affect sedimentation velocity.
V s
= d 2 (
 p
9

 l
) g drag force
Where:


V s l
= settling velocity* of a falling sphere.
d = diameter of the sphere.
p
= density of sphere.
= density of the liquid medium.
 = viscosity of liquid medium.
g = gravitational (centrifugal) force.
*V s is also known as “terminal velocity” (V t
) gravitational force
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 The mathematical ratio of sedimentation velocity (v t
) 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 = v g t v t
Where:
= terminal velocity of a falling sphere (cm/sec).
g = gravitational (centrifugal) force (cm/sec 2 ).
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1.

Cell-free extract and supernatants are centrifuged at progressively higher speeds and longer times.
2.

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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12

Sample
Low
Density
20% sucrose
(1.08 g/mL)
High
Density
Initial
65% sucrose
(1.32 g/mL)
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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.
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 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.



Alanine transaminase
Aspartate transaminase
Alkaline phosphatase
• Activities of one or more enzyme are measured in blood serum.

• Elevated levels indicate liver function abnormalities or damage.

Gamma-glutamyl transferase
Lactate dehydrogenase


Creatine kinase
Troponin
• Elevated serum levels indicate recent heart (attack) damage.
16

Succinate dehydrogenase:


Membrane-bound enzyme of mitochondrion.
Oxidizes succinate to fumarate in TCA Cycle.
C
I
HCH
I
OO
-
HCH
I
C OO
-
Succinate
Dehydrogenase
FAD FADH
2
C
I
CH
II
OO
-
HC
I
C OO
-
Respiratory
Electron
Transport
UQH
2
UQ DCPIPH
2
(Colorless)
DCPIP
(Blue)
O
2
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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.
18

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:
G6PDH:
Fructose-6P
↔
Glucose-6P
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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