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UNIVERSITY OF PÉCSMEDICAL SCHOOL

www.medschool.pte.hu

BIOPHYSICS 2.2015 11th MarchDr. Beáta BugyiDepartment of Biophysics

SEPARATION TECHNIQUES:

� SEDIMENTATION� ELECTROPHORESIS

Applicationssedimentation: hematology – blood fractionation

1000 g, 5- 10 min

PLASMA (55 %)

WHITE BLOOD

CELLS, PLATELETS

(< 1%)

RED BLOOD CELLS

(45 %)

Example: complete blood count

Applicationselectrophoresis of blood serum

Forrás: http://bestpractice.bmj.com/best-practice/monograph/179/diagnosis/step-by-step.html

Example: Healthy (A, C) and multiple myeloma (plasma cell myeloma, myelomatosis, or

Kahler's disease, plasma cell cancer) patients’ (B, D) electrophoretogram Biophysical aspects

� MOVEMENTMOVEMENTMOVEMENTMOVEMENT OF OF OF OF PARTICLESPARTICLESPARTICLESPARTICLES ININININ FORCEFORCEFORCEFORCE FIELDSFIELDSFIELDSFIELDS

� SEDIMENTATION

movement of particles in centrifugal force field

� ELECTROPHORESIS

movement of particles in electric force field

Sedimentation, electrophoresis

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Workflow

� sample: particle suspension (blood-, urine, bone

marrow sample, cells, cellular components,

proteins, nucleic acids…)

� instrument: laboratory centrifuge/electrophoretic

apparatus

� measured quantities: optical signals (absorption,

light scattering, fluorescence emission)

� information/result: size, shape, molecular weight,

density, isoelectric point, nucleotide composition

(physical, chemical, biological parameters)

� SEDIMENTATIONSEDIMENTATIONSEDIMENTATIONSEDIMENTATION� movement of particles in centrifugal force field

� ANALYTICALANALYTICALANALYTICALANALYTICAL ULTRACENTRIFUGATIONULTRACENTRIFUGATIONULTRACENTRIFUGATIONULTRACENTRIFUGATION• determination of the mass and size of particles

� SEDIMENTATION VELOCITY METHOD� SEDIMENTATION EQUILIBRIUM METHOD

� PREPARATIVEPREPARATIVEPREPARATIVEPREPARATIVE ULTRACENTRIFUGATIONULTRACENTRIFUGATIONULTRACENTRIFUGATIONULTRACENTRIFUGATION• separation of particles

� DIFFERENTIAL CENTRIFUGATION» on the basis of mass and size

� GRADIENT CENTRIFUGATION» on the basis of density

Sedimentation

��������� �������

� ����������

��: densitymedium < � ∶densityparticle � ���1 �

����

��

������� � �� � ���

��������� � ��

� ���������� � � � ��

SEDIMENTATION (SETTLING) VELOCITY OF A

PARTICLE

mass

shape

density

gravitational

acceleration

force field

�

Movement of particles in the gravitational field ofEarth

�!"

��

!"

6$%&

�� � � ���������� ' ��������� ' �������

� � 9.81�

+,

Movement of particles in the gravitational field ofEarth

� Estimate how long does it take to settle a protein by 1 cm in

the gravitational field of Earth.

A) few days

B) months

C) C) C) C) hundredshundredshundredshundreds of of of of yearsyearsyearsyears - �+

�~100�+~3002345+

� ��

���1 �

��

��

characteristic to the force

field, not to the particle

HOW?

CAN WE ACCELERATE THE

SEDIMENTATION OF PARTICLES?

FORCE FIELD > DIFFUSION

WHY?

GRAVITATIONAL FORCE < DIFFUSION!

�~106,7!�, ~1060089

:,

! � 1.38 ∗ 106,<=

>, " � 273A,

� � 9.818

:9, + � 106,�

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Instrument – laboratory centrifuge other centrifuges...

20 G centrifuge (NASA Ames Research Center, California)

���������

���������B����� �C

������� � ���DE � ����D

E

��������� � ��

��B����� �C � ��DE � ���DE

SEDIMENTATION (SETTLING) VELOCITY OF A

PARTICLE

�

axis of rotation

F � 2π�

angular frequency

Movement of particles in the centrifugal force field5F,instead of �

� ���1 �

����

�5F,

mass

shape

density

centrifugal

acceleration

force field

rotation

The velocity decreases, as the radius decreases.

�� � ��B����� �C ' ��������� ' �������

��: densitymedium < � ∶densityparticle

�!"

��

!"

6$%&� � 9.81

�

+,

Movement of particles in the centrifugal force field5F,instead of �

4HIJ �4KILMHNOPQRJ�5F

,�

��0.0115S,

�

5 � 10T�

RELATIVE ACCELERATION (RCP)

GRAVITATIONAL ACCELERATION: g = 10 m/s2

change in velocity: 36 km/h within one second

Generally humans can tolerate 3-5*g

HIGH speed of rotation

(ultracentrifuge)

N ~ 100.000 rpm (100x)

��BC � U.U��. ���*g (10.000x!! = 1002)

the protein

- �+

�~10V+~3WXY5+

LOW speed of rotation

N ~ 1000 rpm

��BC � UU�*g

the protein

- �+

�~10Z+~32345+

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�X[\]\-2 ��3]XT\-2

�X5T3�\3]^

SEDIMENTATION MOBILITY (S: Svedberg, Theodore Svedberg)

_ ��

5F,�

��1 �`�`�

�

Svedberg:

� physical quantity describing the sedimentation properties of particles

� equals to the sedimentation velocity of a particle in a unit force field

� [Svedberg] = 1S = 10-13s (SI unit)

Sedimentation mobility

�!"

�~

!"

6$%&

Sedimentation mobility

particlesedimentation mobility

(S, 10-13 s, 20oC)

molecular weight

(Dalton)

O2 0.12 32

glucose 0.18 180

insulin monomer 1.5 6000

ribonuclease 1.75 13.683

lyzosim 2.03 17.200

insulin 1.84 24.430

serum albumin 4.3 68.460

catalase 10.7 250.000

tobacco mosaic virus 175 31.340.000

_ � 175_ � 175 ∗ 1060<s

5F, � 10c�

+,� 100.000 ∗ �

� � _5F, � 175 ∗ 1060<s ∗ 10c�

+,�

� Ud.e ∗ U�6f�

g�hi

j�

Analitical centrifugationsedimentation velocity method

distance from the axis of rotation (r)

radial distribution of particle

concentration in time c�5, -�

mass

Boltzmann constant

temperature

T�5, -� →_

~��1 �

`�`�

!"

diffusion coefficient

(has to be measured!)

high F

sedimentation

nT

n-�

n,T

n5,'1

5

nT

n5� _F,o5

nT

n5' 2Tp

timetimetimetime

���, ��

Mass determination, combined with

diffusion measurements!

~ speed of

centrifugation

concentration~absorption

~ fluorescence

time evolution of radial concentration distribution

Analitical centrifugationsedimentation equilibrium method

radial distribution of particles in equilibrium

(Boltzmann distribution): T�5�

T�5�~��1 �

`�`�F,

!"

mass

Boltzmann constant

temperature

~ speed of

centrifugation

T�5� � T�3qr��1 �

`�`�F,

!"�5, � 5�

,

2�

diffusiondiffusiondiffusiondiffusion sedimentationsedimentationsedimentationsedimentation

low Fsedimentation velocity = 0

equlibrium between diffusion & sedimentation

����

Mass determination!

concentration~absorption

~ fluorescence

distance from the axis of rotation (r)

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Preparative ultracentrifugationdifferential centrifugation

SEPARATION OF TISSUE

COMPONENTS ON THE BASIS OF

THEIR SEDIMENTATION

PROPERTIES

sequential centrifugations� �

��U �����

�5F,

� � �� → � � �

DENSITY GRADIENT

sample

Preparative ultracentrifugationgradient centrifugation

SEPARATION BASED ON DENSITY

�� = density of the medium

�= density of the particle

� ELECTROPHORESISELECTROPHORESISELECTROPHORESISELECTROPHORESIS� movement of particles in electric field

� FREE ELECTROPHORESISFREE ELECTROPHORESISFREE ELECTROPHORESISFREE ELECTROPHORESIS� GELELECTROPHORESISGELELECTROPHORESISGELELECTROPHORESISGELELECTROPHORESIS

• POLYACRYLAMIDEPOLYACRYLAMIDEPOLYACRYLAMIDEPOLYACRYLAMIDE GELELECTROPHORESISGELELECTROPHORESISGELELECTROPHORESISGELELECTROPHORESIS –––– proteinsproteinsproteinsproteins

� NATIVE GELELECTROPHORESIS» separation on the basis of size, shape and charge

� DENATURING GELELECTROPHORESIS» separation on the basis of size

� ISOELECTRIC FOCUSING» separation on the basis of isoelectric point

� 2D ELECTROPHORESIS» separation on the basis of isoelectric point and size

• AGAROSEAGAROSEAGAROSEAGAROSE GELELECTROPHORESISGELELECTROPHORESISGELELECTROPHORESISGELELECTROPHORESIS –––– nucleicnucleicnucleicnucleic acidsacidsacidsacids» separation on the basis of size

Electrophoresis Instrument – electrophoretic apparatus

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��������� � ��

�s�C�� � tu � vBu

positivepositivepositivepositive electrode

nnnnegativeegativeegativeegative electrode

negative particles

anions

positive particles

cations

po

ten

tia

ldif

fere

nce ���������

�s�C��

ELECTROPHORETIC VELOCITY OF THE

PARTICLE

charge

Movement of particles in electric fields

� �w3

�x

shape

electric

field strength

�� � �s�C�� ' ���������

Electrophoretic mobility

u:

� physical quantity describing the electrophoretic properties of particles

� equals to the electrophoretic velocity in a unit force field

� [u] = m2*V-1*s-1

�X[\]\-2 ��3]XT\-2

�X5T3�\3]^

ELECTROPHORETIC MOBILITY

��

u�vB

�

Gelelectrophoresis (GE)Matrix, gel

� polyacrylamide (PAGE: polyacrylamide gel electrophoresis), agarose based 3D

cross-linked polymer lattice

� based on the pore size of the mesh network

� macromolecules are separated according to their size

acrylamide

(%)

linear

separation

range

(kda)

agarose

(%)

DNA size

(base pair)

5 57-212 0.2 5000-40000

7.5 36-94 0.4 5000-30000

10 16-68 0.6 3000-10000

15 12-43 0.8 1000-7000

1 500-5000

2 200-1500

3 100-1000

concentration ↑

pore size↓

separation range↓

large

small

Native and denaturing gelelectrophoresis

NATIVE DENATURING (SDSPAGE)

REDUCINGREDUCINGREDUCINGREDUCING, , , , DENATURINGDENATURINGDENATURINGDENATURING ENVIRONMENTENVIRONMENTENVIRONMENTENVIRONMENT

� SDS (sodium dodecyl sulfate, anionic

detergent)� negativenegativenegativenegative chargechargechargecharge

� β−MEA (β-mercaptoethanol) �

disruption of disulfid bridges

� heat treatment�denaturationdenaturationdenaturationdenaturation

separationseparationseparationseparation, , , , accordingaccordingaccordingaccording totototo::::

SIZESIZESIZESIZE

independent of native charge

independent of native shape

NONREDUCINGNONREDUCINGNONREDUCINGNONREDUCING, , , , NONDENATURINGNONDENATURINGNONDENATURINGNONDENATURING

ENVIRONMENTENVIRONMENTENVIRONMENTENVIRONMENT

� macromolecule in its native state

separationseparationseparationseparation, , , , accordingaccordingaccordingaccording totototo::::

SIZESIZESIZESIZE

SHAPESHAPESHAPESHAPE

CHARGECHARGECHARGECHARGE

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Isoelectric focusing

START: inhomogeneous distribution

pH pH pH pH gradientgradientgradientgradient

pH pH pH pH gradientgradientgradientgradient

END: distribution according to isoelectric point

positive chargenegative charge

separationseparationseparationseparation, , , , accordingaccordingaccordingaccording totototo::::

ISOELECTRICISOELECTRICISOELECTRICISOELECTRIC POINTPOINTPOINTPOINT

2D electrophoresis

1D: pH gradient

isoelectric focusing

1D: SDSPAGE

separation according to size

2D electrophoresis

sample from mouse brain, source: www.bio-rad.com

ISOELECTRIC POINT

SIZE

Gel staining, quantification

Coomassie blue

protein

silver stain

protein

Fluorescent dye

protein, nucleic

acid

ethidium

bromide

nucleic acid

sensitivity 5 - 25 ng 0.25 - 1 ng 0.25 – 8 ng 0.5 to 5.0 ng

ease of use

coomassie dye binds to

basic and hydrophobic

residues of proteins

silver ions interact and

bind with with

carboxylic acid groups (

Asp and Glu),

imidazole (His),

sulfhydryls (Cys), and

amines (Lys).

simply dye-binding

mechanisms DNA interchalator

appearanceabsorbance

blue

absorbance

dark brown

fluorescence

emission

fluorescence

emission

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Applicationsquantitative and qualitative analysis

qualitative analysis, calculation of:

� SIZE (MOLECULAR WEIGHT, BASE PAIRS)

� AMOUNT

� ISOELECTRIC POINT

quantitative analysis, separation according to:

� SIZE (MOLECULAR WEIGHT, BASE PAIRS)

� CHARGE

� SHAPE

� ISOELECTRIC POINT

Standards

Applicationsqualitative analysis – determination of molecular mass

http://www.biochem.arizona.edu/classes/bioc462/462a/NOTES/Protein_Properties/protein_purification.htm

1. known molecular weight proteins (standard)

2. unknown molecular weight proteins

Applicationselectrophoresis of blood serum

protein normal value

Albumin Albumin Albumin Albumin 55.8 55.8 55.8 55.8 ---- 65% 65% 65% 65%

α1 α1 α1 α1 globulinglobulinglobulinglobulin 2.2 2.2 2.2 2.2 ---- 4.6 % 4.6 % 4.6 % 4.6 %

α2 α2 α2 α2 globulinglobulinglobulinglobulin 8.2 8.2 8.2 8.2 ---- 12.5 % 12.5 % 12.5 % 12.5 %

β(1+2) β(1+2) β(1+2) β(1+2) globulinglobulinglobulinglobulin 7.2 7.2 7.2 7.2 ---- 14.2 % 14.2 % 14.2 % 14.2 %

γ γ γ γ globulinglobulinglobulinglobulin 11.5 11.5 11.5 11.5 ---- 18.8 % 18.8 % 18.8 % 18.8 %

amount

electrophoretic

mobility

ELECTROPHORETOGRAM

www.aok.pte.hu

Sedimentation, electrophoresiskeywords� MovementMovementMovementMovement of of of of particlesparticlesparticlesparticles inininin forceforceforceforce fieldsfieldsfieldsfields ((((centrifugalcentrifugalcentrifugalcentrifugal, , , , electricelectricelectricelectric))))

� AnalyticalAnalyticalAnalyticalAnalytical and and and and preparatvepreparatvepreparatvepreparatve ultracentrifugationultracentrifugationultracentrifugationultracentrifugation techniquestechniquestechniquestechniques

� FreeFreeFreeFree---- and and and and gelgelgelgel----electrophoresiselectrophoresiselectrophoresiselectrophoresis

� NativeNativeNativeNative, , , , denaturingdenaturingdenaturingdenaturing electrophoresiselectrophoresiselectrophoresiselectrophoresis, , , , isoelectricisoelectricisoelectricisoelectric focusingfocusingfocusingfocusing, 2D , 2D , 2D , 2D

electrophoresiselectrophoresiselectrophoresiselectrophoresis

� QualitativeQualitativeQualitativeQualitative and and and and quantitativequantitativequantitativequantitative analysisanalysisanalysisanalysis