University of Washington - Department of Chemistry

Chemistry 453

Homework Assignment 4: due at 5 pm on Fr

Problems from the book


24.7





Sedimentation of chromatin (this first problem is in essence problem 24.29)

Chromatin is the complex of DNA and proteins (mostly histones) found in all eukaryotic
cells. The fundamental repeating unit of chromatin is the nucleosome particle. The
properties of the DNA and the protein in nucleosome particles were determined using a
combination of velocity sedimentation, dynamic light scattering, and gel electrophoresis.

Dynamic light scattering studies determined the diffusion coefficient of the nucleosome
particle in solution at 293K to be 4.37x10
-7
cm
2
/s. In the same solution velocity
sedimentation performed at 18,100 revolutions per minute, obtained the following data:

Time (minutes) Boundary Position r (cm)
0 4.460
80 4.593
160 4.713
240 4.844

Gel electrophoresis showed that the DNA molecule associated with a single nucleosome
protein complex is 200 base pairs in length.

a) What is the molecular weight of the nucleosome particle? Assume the solution has a
density of 1.02g/cm
3
and the specific volume of the nucleosome particle is 0.66cm
3
/g.

By plotting ln(r) versus t

The slope is
( )( )
( )
2
6 1
2 6 1 12
2
1
5.76 10 sec 60sec/ min
5.76 10 sec 1.60 10 sec 15.8
2 18,100min
s s S

= = = =

Then take the usual relationship for s:
D N
T k V M
f N
V M
s
A
B
A
) 1 ( ) 1 (
2 2 2 2

=

=
Rearrange it and solve for the molecular weight:
D V
T sk
N M
B
A
) 1 (
2
2

=

( )( )( )( )
( )( ) ( )( )
23 1 16 12
5
7 2
6.02 10 1.38 10 / 1.60 10 sec 293
2.73 10 /
1 0.66 1.02 4.37 10 /
mole ergs K K
g mole
cm s

= =



b) Assuming the nucleosome particle is spherical, calculate its Stokes radius (assume the
viscosity of the solution is 0.01 gm cm
-1
s
-1
).

For a spherical particle:
R
T k
D
B
6
=
R
T k
R
B
6
=
cm x
s cm x cmxsx g
K K ergs x
R
7
2 7
16
10 91 . 4
/ 10 37 . 4 / 01 . 0 6
293 / 10 38 . 1

= =

c) Each base pair in DNA is separated from the adjacent base pairs by about 3.4x10
-10
m;
so a piece of DNA 200 base pairs long is approximately 68x10
-7
cm in length. Based on
ln(r) versus t
1.48
1.5
1.52
1.54
1.56
1.58
1.6
0 5000 10000 15000 20000
time (seconds)
l
n
(
r
)
this result, and the result from part b, comment on how tightly packed the DNA is in the
nucleosome.

Because the length of the DNA is much greater than the diameter of the nucleosome
particle we assume the DNA must be tightly folded in the nucleosome.

d) How are the proteins and DNA packed in the nucleosome? To answer this question,
assume the eight proteins form a unhydrated spherical complex with specific volume 0.74
cm
3
/g. Calculate the radius of this hypothetical protein sphere.


e) Assume the 200 base pair DNA behaves as a random coil polymer. Calculate the root
mean square (rms) end-to-end distance. Comparing the protein sphere radius with the rms
end-to-end distance for the DNA, is most of the DNA packed outside or inside the protein
core of the nucleosome? Explain.

cm x cmx x N l R
DNA
7 8 2
10 8 . 4 200 10 4 . 3

= = =
Because R R
DNA protein
2
>> , the DNA occupies a larger volume than the protein and is
thus likely wrapped around the protein in the nucleosome.



These next few problems are taken from the old book I used until last year.


Sedimentation of DNA
The sedimentation coefficient of a certain DNA in 1M NaCl at 20C was measured by
boundary sedimentation at 24,630 rpm. The following data were recorded:

Time (min): Distance x of boundary from center (cm)
16 6.269
32 6.351
48 6.438
64 6.517
80 6.605
96 6.681

a) Plot log x vs time and calculate the sedimentation coefficient
( )( )
( )( )
( )
5 5
1/ 3
2
23 1
Pr
2.73 10 / 200 660 / 1.38 10 /
138,000 / 0.74 /
3 3
3.5 10
4 4
6.02 10
protein
A
Weight Histone oteinComplex Weight Nucleosome Weight DNA
gm mole base pairs gm molebase pairs gm mole
g mole cc g
MV
R
N
mole

=
=


= = =

7
cm
b) The partial specific volume of the sodium salt of DNA is 0.556 cm
3
/g. The viscosity
and density of the 1M NaCl solution and of water are 1.104 cP, 1.04 g/cm
3
and 1.005 cP,
respectively. Calculate s
20,w
for the DNA

a) Plot ln(r) versus time (in seconds)

The slope of the plot is
2
s=5.5x10
-6
sec
-1
, therefore:

S s x
s x x
s x
s 2 . 19 10 9 . 1
60 min/ min 630 , 24 2
10 5 . 5
12
1
1 6
= = =

b)
( )
( )
S
x
x
S
V
V
s s
w
w
w
2 . 22
104 . 1 556 . 0 1
998 . 0 556 . 0 1
005 . 1
104 . 1
2 . 19
1
1
2
, 20 2
, 20
, 20
=

Sedimentation of bacteriophage T7
For bacteriophage T7, the following data were obtained
s
0
20,w
=453S
D
0
20,w
=6.03x10
-8
cm
2
/s
V
2
=0.639 cm
3
/g (this is the partial specific volume)

a) Calculate the molecular weight of the phage particle
b) Phosphorous and nitrogen analysis of the bacteriophage show that 51.2% by weight of
the phage is DNA. Calculate the molecular weight of T7 DNA. Each bacteriophage
contains one DNA molecule.

a) ( )D V
RT
M
f
V
N
M
s
A
2
2 2 2
1
1

=
6.5: DNA Sedimentation
1.83
1.84
1.85
1.86
1.87
1.88
1.89
1.9
1.91
0 2000 4000 6000 8000
ti me (sec)
l
n
(
r
)
Series1
Hence:
( ) ( )
mole g x
s cm x x cm g gx cm
K Kxmolex ergs x sx x
D V
sRT
M / 10 05 . 5
10 03 . 6 / 998 . 0 / 639 . 0 1
293 / 10 31 . 8 10 453
1
7
1 2 8 3 3
7 13
2
2
=

b) The molecular Weight of T7 DNA is:

mole g x molex g x / 10 58 . 2 512 . 0 / 10 05 . 5
7 7
= =



Sedimentation of virus head group
T4 is a large spherical virus with an approximately spherical head group which contains
DNA. These head particles have the following characteristics:

s
0
20,w
=1025 S
D
0
20,w
=3.6x10
-8
cm
2
/s
V
2
=0.605 cm
3
/g (this is the partial specific volume)
Calculate:
a) The molecular weight of the head group
b) The volume of the head group from the partial specific volume (assume no
hydration)
c) The frictional coefficient of the head group from the diffusion coefficient
d) The volume of the head group from the frictional coefficient in part c and Stokes
equation.
e) How many g of water of hydration are there, per g of protein?


a) ( )D V
RT
M
f
V
N
M
s
A
2
2 2 2
1
1

=
Hence:
( ) ( )
mole g x
s cm x x cm g gx cm
K Kxmolex ergs x sx x
D V
sRT
M / 10 76 . 1
10 60 . 3 / 998 . 0 / 605 . 0 1
293 / 10 31 . 8 10 1025
1
8
1 2 8 3 3
7 13
2
2
=

b) molecule cm x
mole molecules x
g cm molex g x
N
V M
V
A
/ 10 77 . 1
/ 10 02 . 6
/ 605 . 0 / 10 76 . 1
3 16
23
3 8
2 2
= = =

c)
16
6
8 2
1.38 10 / 293
1.12 10 /sec
3.60 10
kT x erg Kx K
f x g
D x cm s

= = =

d) R f 6 =
hence: cm x
scm g x
s g x f
R
6
6
10 91 . 5
/ 01 . 0 6
/ 10 12 . 1
6

= = =



And therefore:
3 16
3
10 65 . 8
3
4
cm x
R
V

= =



The volume of water per protein is 6.88x10
-16
cm
3


e) Because the partial specific volume of water is about 1cm
3
/gram, we have 6.88x10
-16
g
of water hydrating a protein molecule. We can convert this to grams of water hydrating
each gram of protein:

gprotein gwater n moleprotei molecules x x
n moleprotei gprotein x
ecule proteinmol gwater x
/ 35 . 2 / 10 02 . 6
/ 10 76 . 1
/ 10 88 . 6
23
8
6
=

Text Problem
Describe (a) one method that can be used to determine the absolute molecular weight of a
protein and (b) how an equilibrium binding constant can be determined by gel
electrophoresys

a) Sedimentation and diffusion, or sedimentation equilibrium
b) If the kinetics of the equilibrium is slow compared to electrophoretic rates, a product
can be separated from the reactants. The relative amount of material in the gel bands
provides the concentrations necessary to obtain the equilibrium constant.