Laboratory Activity Ten - PowerPoint Presentation

Slide1

Laboratory Activity Ten

Centrifugation Techniques

& the Isolation ofCauliflower Mitochondria

1Slide2

Purposes of Lab 10

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.2Slide3

Introduction to Centrifugation

Definition:A technique (or process) where centrifugal force is used to separate mixtures of particles or substances on the basis of their differential densities.Theory: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.3

(RPM)

(Centrifugal Force)Slide4

Generation of Centrifugal Force

4

(Radius, r)

in cm

RCF = (1.12 x 10

-5

) x (r, cm) x RPM

2

(r = 1 cm)

(r = 5 cm)

(r = 10 cm)



Note:

RCF (Relative Centrifugal Force) is expressed as no. of x’s gravity.Slide5

Applications for Centrifugation

5

Industrial DecanterSlide6

Applications for Centrifugation

6

Refrigerant

Vacuum

Drive

Motor

Armored

PlateSlide7

Densities of Biological Materials

7

Biological MaterialBuoyant

Density (g/cc)

RNA

DNA

Ribosomes

Nuclei

Chloroplasts

Mitochondria

Endoplasmic

reticulum

Lipid bodies

1.90

1.70

1.60

1.32

1.21

– 1.24

1.18 – 1.20

1.11 – 1.12

0.96Slide8

Stoke’s Law & Centrifugation

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.8

d2 (p

–



l

) g

9



V

s

=

Where:

V

s

= 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.

*

V

s

is also known as “terminal velocity” (

V

t

)

drag force

gravitational

forceSlide9

Sedimentation Coefficients

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.9

s =

v

t

g

Where:

v

t

= terminal velocity of a falling sphere (cm/sec).

g = gravitational (centrifugal) force

(

cm/sec

2

).Slide10

Sedimentation Coefficients of Sample Biological Particles

10

Particle

Svedberg Unit

Cytochrome c

tRNA

Eukaryotic Ribosomes

Large SU

Small SU

Prokaryotic Ribosomes

Large SU

Small SU

Tobacco Mosaic Virus

Mitochondria

1.7S

4S

80S

60S

40S

70S

50S

30S

200S

20,000-60,000SSlide11

Main Types of Centrifugation Techniques

Differential CentrifugationCell-free extract and supernatants are centrifuged at progressively higher speeds and longer times.Density Gradient CentrifugationCell-free extract is centrifuged through a medium whose density gradually increases toward the bottom of the centrifuge tube.

Rate Zonal – sample is centrifuged until most dense component approaches bottom of centrifuge tube.Isopycnic – sample is centrifuged until all components reach their equilibrium buoyant density.

11Slide12

A Closer Look atDifferential Centrifugation

12Slide13

Density Gradient Centrifugation

13

Sample

Low

Density

High

Density

Initial

65% sucrose

(1.32 g/mL)

20

% sucrose

(1.08 g/mL)Slide14

Density Gradient Centrifugation

14

Rate Zonal Centrifugation

D

ensity of medium <

densities of particles.

Particles with similar densities move as bands towards

bottom.

Particles

will/may pellet out or mix at bottom.

Isopycnic

Centrifugation

Maximum d

ensity

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

.Slide15

Preparation of Cauliflower Mitochondria

(To be performed by TA’s)15

“Shave” off

approximately 15 g

of floret tissue.Slide16

Marker Enzymes

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 transaminaseAlkaline phosphataseGamma-glutamyl transferaseLactate dehydrogenaseCreatine kinaseTroponin

16

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.Slide17

Marker Enzymes in BIOSC 434

Succinate dehydrogenase:Membrane-bound enzyme of mitochondrion.Oxidizes succinate to fumarate in TCA Cycle. 17

C

I

HCH

I

HCH

I

C

OO

-

OO

-

C

I

CH

II

HC

I

C

OO

-

OO

-

Succinate

Dehydrogenase

FAD

FADH

2

UQ

UQH

2

DCPIPH

2

(Colorless)

DCPIP

(Blue)

Respiratory

Electron

Transport

O

2Slide18

Marker Enzymes in BIOSC 434

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.18Slide19

Marker Enzymes in BIOSC 434

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.19

PGI:

Fructose-6P

↔

Glucose-6P

G6PDH

:

Glucose-6P

+ NAD

+ + H

+ ↔ 6-Phosphogluconate + NADHSlide20

Specific Activities for Lab 10

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.20Slide21

Questions or Comments

21