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Ch 24pages 636-643
Lecture 11 – Equilibrium centrifugation
Sedimentation can be used very effectively to separate, purify and analyze all kind of cellular components
It can be understood using the mechanical analogy with flow under gravitation. The steady state velocity at which a particle move under centrifugation is determined by the balance between the angular velocity at which centrifugation occurs and the opposing buoyancy and frictional forces.
Summary of Lecture 10
0 fbg FFF
rsr
f
Vm
dt
drv 2221
We have introduced the sedimentation coefficient:
Summary of Lecture 10
f
Vms
21
Its dimensions are sec, but a more convenient unit is the Svedberg: S=10-13 s
Values for s are usually referred to pure water at 293K=20oC. Under these conditions, the sedimentation coefficient is indicated as follows:
R
Vms
w
ww
,20
,202,20 6
1
A sedimentation coefficient measured under other conditions, i.e. in a buffered aqueous solution b and/or at another temperature T can be related to standard conditions by the equation
Summary of Lecture 10
Boundary sedimentation is an equilibrium technique that can be used to separate and analyze macromolecules by sedimentation.
R
Vms
w
ww
,20
,202,20 6
1
w
wbTw
V
Vss
,202
,202,,20
1
1
A macromolecular solution subjected to a centrifugal field will quickly attain a steady state condition in which transport of solute mass occurs at constant velocity and a concentration gradient will be generated
If we spin very fast, eventually the entire macromolecular population will deposit at the bottom of the tube, at a rate that depends on the centrifugal speed and the density and viscosity of the solvent
If we do not spin too fast, centrifugation and diffusion will balance each other out so that the system will attain equilibrium, at which point net transport will cease and transport velocity is zero
Equilibrium Centrifugation
This is because transport by centrifugation and by diffusion will oppose and balance each other: centrifugation will generate a density gradient, and diffusion will try and eliminate such gradient
The rate at which equilibrium is reached depends on kinetic properties (diffusion coefficient, angular velocity etc)
The equilibrium state does not
The concentration at equilibrium is only determined by thermodynamic properties of the system and not by sedimentation coefficients, diffusion etc
Equilibrium Centrifugation
The equilibrium concentration can be derived by considering Boltzmann’s distribution. Let us consider again the analogy with gravitation. Molecules in a gravitational field will have different energy depending on whether they are higher or lower; the distribution of molecules in different energy levels is given by Boltzmann’s expression:
Equilibrium Centrifugation
kTEEP
Pji
j
i /exp
The probability ratio is equivalent to the ratio of concentrations; if we express the energy per mole instead of per particle by multiplying by Avogadro’s number, we find:
RTEEC
Cji
j
i /exp
Let us return to centrifugation; the centrifugal acceleration for a centrifuge spinning with angular speed is r, the force acting on the particle is
Equilibrium Centrifugation
Therefore, the energy is:
rVm 22 )1(
2
)(22
02
0
rmmrdrmmdrFFrU lcentrifugabuoyancy
222 )1(
2rV
mE
Substituting this expression we immediately find the expression for the concentration as a function of the axial distance r:
Equilibrium Centrifugation
222 )1(
2rV
mE RTEE
C
Cji
j
i /exp
20
2222
0 2
1)(ln rr
RT
VM
C
rC
Another derivation re-introduces the chemical potential. The condition of equilibrium requires that the free energy, i.e. the system’s chemical potential, which is the sum of the chemical and centrifugal potential, is minimum. If you think about how centrifugation works, what we would like to know is really the concentration profile with respect to the radius r not as a function of time (as in non-equilibrium centrifugation), but rather at equilibrium. We can then impose that the derivative of the chemical potential with respect to r is 0 at equilibrium. In lecture 6, we have introduced the chemical potential in analogy to the classical concept of force as of a potential gradient, to express differences in free energy that induce diffusion
Equilibrium Centrifugation
If the concentration of solute C2 is a function of x, the chemical
potential has the general form
Equilibrium Centrifugation
)(ln)( 2022 xCRTGxG
A difference in chemical potential exercises a force on the solute molecules, and the force that induces solute flow is related to the chemical potential by the diffusion equation. In the case of centrifugation, we have to add a term that describes the centrifugal field, so that the total chemical potential of the system is:
)()(ln0 rUrCRTGsolutelcentrifugachemtot
Here C is the solute concentration at position r in the centrifuge tube and U is the centrifugal potential at position r in the centrifuge tube. At equilibrium:
Equilibrium Centrifugation
)()(ln0 rUrCRTGsolutelcentrifugachemtot
0 lcentrifugachemicaltotal
dr
d
dr
d
0)()(ln0
dr
rdU
dr
rCdRT
dr
dGsolute
We can substitute for dU/dr the expression for the centrifugal force provided in the previous lecture (remember, a force is a gradient of a potential), then multiply by Avogadro’s number to obtain the centrifugal potential per mole of solute (which is what the chemical potential will be expressed in) and equate the derivative of the concentration with the derivative in centrifugal potential to obtain:
Equilibrium Centrifugation
)()(ln0 rUrCRTGsolutelcentrifugachemtot
20
ln ln ( )( )B buoyancy centrifugal
A
d C r d C rRT dU rk T F F m m r
N dr dr dr
Integrating that equation:
Equilibrium Centrifugation
)()(ln0 rUrCRTGsolutelcentrifugachemtot
20
ln ln ( )( )B buoyancy centrifugal
A
d C r d C rRT dU rk T F F m m r
N dr dr dr
22
2 20 2 2 2 20 0
0
1ln
2 2B
M Vm mCr r r r
C k T RT
2
2 2 2 20
0
1ln
2
M VCr r
C RT
Example : Calculate the weight of carboxy-hemoglobin (cHb) using the following data obtained from an equilibrium centrifugation experiment
Equilibrium Centrifugation
At r=4.61 cm C(cHb)=1.220 weight %At r=4.56 cm C(cHb)=1.061 weight %T=293.3K spinning at 8703 revolutions/minuteThe specific volume of cHb is 0.749 cm3/g.The density of water is 1g/cm3
22
02
2
02
1
/ln2
rrV
CCRTM
Equilibrium Centrifugation
At r=4.61 cm C(cHb)=1.220 weight %At r=4.56 cm C(cHb)=1.061 weight %T=293.3K spinning at 8703 revolutions/minuteThe specific volume of cHb is 0.749 cm3/g.The density of water is 1g/cm3
7
23 3 1 2 2
2 8.31 10 / 293.3 ln 1.22 /1.06170,900 /
1 0.749 / 1 / 2 8703min min/ 60 (4.61 ) (4.56 )
x x ergs molexKx Kxg mole
cm gx g cm x s cm cm
22
02
2
02
1
/ln2
rrV
CCRTM
This equation means that when a solution reaches equilibrium in a centrifugal field, generated by spinning the sample at an angular frequency , a concentration gradient will be generated of the shape given above
This experiment can be used to measure macromolecular masses or separate components of a mixture
A plot of lnC vs. r2 is a straight line with slope proportional to M. Notice that this method provides absolute molecular weight, while electrophoresis, for example, only provides relative molecular weights
Equilibrium Centrifugation
2
2 2 2 20
0
1ln
2
M VCr r
C RT
Example : Consider the centrifugal separation of two gases with molecular weights of 349 g/mole (1) and 352 g/mole (2). How fast do you have to spin the sample to enrich molecule 1 to a level of 1% at r=3cm if its level is 0.7% at r=10cm and T=273K.
Equilibrium Centrifugation
20
2222
0, 2
1ln rr
RT
VM
C
C
i
i
Set the buoyancy correction to 1 for gases and subtract the two expressions for C2 and C1 to obtain:
20
2212
10,2
0,12
2ln rr
RT
MM
CC
CC
Then solve for 2
Equilibrium Centrifugation
10,2
0,12
20
212
2 ln2
CC
CC
rrMM
RT
7
2 7 2
2 2 2
2 8.31 10 / 273 0.01ln 5.92 10
0.007352 / 349 / 10 3
x x ergs molexKx Kx s
g mole g mole cm
37.69 10 /x radians s
3 11.22 10x s
A type of equilibrium centrifugation involves spinning of a concentrated salt solution at very high speed to generate a density gradient (the density of the solution increases with the salt concentration) and has proven very useful in the study of nucleic acids
If a macromolecule is also present, it will form a boundary at a point in the salt gradient where the macromolecules are buoyant
Equilibrium Sedimentation in a Density Gradient
Suppose a solution of a macromolecule (e.g. DNA) also contains a salt such as CsCl
Initially the salt and the DNA have uniform concentrations.
Once the centrifugation has commenced, the salt quickly reaches equilibrium; the concentration of CsCl will reach equilibrium as described by the equilibrium centrifugation equation:
Equilibrium Sedimentation in a Density Gradient
20
22
0, 2
1ln rr
RT
VM
C
C CsClCsCl
CsCl
CsCl
Because of the equilibrium condition, the density of the solution will vary as a function of r, the distance from the spinning axis. Suppose at r’ the solution has a density
Equilibrium Sedimentation in a Density Gradient
20
22
0, 2
1ln rr
RT
VM
C
C CsClCsCl
CsCl
CsCl
1
2V
where is the specific volume of the macromolecule
Equilibrium Sedimentation in a Density Gradient
20
22
0, 2
1ln rr
RT
VM
C
C CsClCsCl
CsCl
CsCl
Equilibrium Sedimentation in a Density Gradient
At r<r’ the density of the solution is less than 1
2V
the DNA “sinks” to the bottom of the tube, being pulled “downward” by the centrifugal force
At r>r’ the density of the solution is greater than 1
2V
At r=r’ the DNA density increases
the DNA ”floats” “upwards” toward the top of the centrifuge
Equilibrium Sedimentation in a Density Gradient
20
22
0, 2
1ln rr
RT
VM
C
C CsClCsCl
CsCl
CsCl
suppose the solution density gradient is roughly linear, with
dr
drr
Vr
'1
)(2
where d/dr is the density gradient of the solution near r’, and is assumed to be a constant in this region, if we do not go very far from r’
Equilibrium Sedimentation in a Density Gradient
20
22
0, 2
1ln rr
RT
VM
C
C CsClCsCl
CsCl
CsCl
Then the condition for equilibrium of the DNA is:
If the solution density in the buoyancy correction is the function of r defined above, then:
dr
drr
Vr
'1
)(2
r
RT
rVM
dr
Cd DNADNADNA 2
2
)(1ln
r
RT
dr
drr
VVM
dr
Cd DNA
DNADNA
DNA 2
2
'1
1ln
Equilibrium Sedimentation in a Density Gradient
20
22
0, 2
1ln rr
RT
VM
C
C CsClCsCl
CsCl
CsCl
Integrate the equation to find, and after some simple calculus:
Although this equation seems similar to the equilibrium centrifugation equation, it has a term (r-r’)2 instead of r2-r’2, so it differs
dr
drr
Vr
'1
)(2
22
''2)'(
)(ln rrr
dr
d
RT
VM
rC
rC DNADNA
DNA
DNA
Equilibrium Sedimentation in a Density Gradient
The logarithm can be removed to obtain:
dr
drr
Vr
'1
)(2
22
''2)'(
)(ln rrr
dr
d
RT
VM
rC
rC DNADNA
DNA
DNA
2
2
''2
exp)'()( rrrdr
d
RT
VMrCrC DNADNA
DNADNA
2
2
'( ) exp
2DNA
r rC r
Equilibrium Sedimentation in a Density Gradient
This is a gaussian with width:
2
2
2
'exp)'(
rr
rCDNA
drdVMr
RT
DNADNA /'2
2
Notice that the standard deviation increases as the salt gradient d/dr decrease; it also depends on the inverse mass, so that the higher the mass, the sharper the gradient
Equilibrium Sedimentation in a Density Gradient
2
2
2
'exp)'(
rr
rCDNA
drdVMr
RT
DNADNA /'2
2
Many macromolecules have buoyant densities sufficiently different that they can be separated by this technique. Hybrid DNA-RNA was discovered using equilibrium centrifugation in a salt gradient, for example, although they differ in mass by very little.