Electric Birefringence of Restriction Enzyme

Fragments of DNA: Optical Factor and Electric

Polarizability as a Function of Molecular Weight

NANCY C. STELLWAGEN,* Department of Chemistry, R-017,

University o f California, S a n Diego, La Jolla, California 92093

Synopsis
T h e electric birefringence of restriction enzyme fragments of DNA has been investigated
as a function of DNA concentration, buffer concentration, and molecular weight, covering
a molecular weight range from 80 to 4364 base pairs (bp) (6 X lo4-3 X lo6 daltons). The
specific birefringence of the DNA fragments is independent of DNA concentration below 20
pg DNA/ml, but decreases with increasing buffer concentration, or conductivity, of the solvent.
At sufficiently low field strengths, the Kerr law is obeyed for all fragments. The electric field
a t which the Kerr law ends is inversely proportional to molecular weight. In the Kerr law
region the rise of the birefringence is accurately symmetrical with the decay for fragments
5 389 bp, indicating an induced dipole orientation mechanism. The optical factor calculated
from a 1/E extrapolation of the high field birefringence data is -0.028, independent of mo-
lecular weight; if a 1/E2extrapolation is used, the optical factor is -0.023. The induced po-
larizability, calculated from the Kerr constant and the optical factor, is proportional to the
square of the length of the DNA fragments, and inversely proportional to temperature.
Saturation curves for DNA fragments 5 161 bp can be described by theoretical saturation
curves for induced dipole orientation. The saturation curves of larger fragments are broad-
ened, because of a polarization term which is approximately linear in E, possibly related to
the saturation of the induced dipole in high electric fields. This “saturated induced dipole”
is found to be 6400 D, independent of molecular weight. The melting temperature of a 216-bp
sample is decreased 6OC in an electric field of 8 kV/cm, because the lower charge density of
the coil form of DNA makes it more stable in an electric field than the helix form.

INTRODUCTION
The mechanism of orientation of DNA in an electric field has been the
subject of many investigations in recent years, with seemingly contradictory
results. Rau and Bloomfield’ found the electric birefringence relaxation
kinetics of T7 viral DNA to be consistent with induced dipole orientation,
but they needed to postulate a field-induced restoring force to explain the
kinetics of the rise of the birefringence. Sokerov and Weill,2 studying
electric birefringence, electric dichroism, and polarized fluorescence in an
electric field of sonicated DNA fragments complexed with the dye acridine
orange, interpreted their results in terms of saturation of the induced dipole
in high electric fields. Hogan et d.,3studying the electric dichroism of DNA
restriction enzyme fragments, interpreted the saturation behavior in terms
* Present address: 1409 Cedar Street, Iowa City, Iowa 52240.

Biopolymers, Vol. 20,399-434 (1981)

Q 1981 John Wiley & Sons, Inc. CCC 0006-3525/81/030399-36$03.60
400 STELLWAGEN

of an apparent permanent dipole moment caused by anisotropic ion flow

about a polyion in an electric field. Houssier and coworker^^?^ interpreted
the electric birefringence saturation behavior of calf thymus DNA and
sonicated fragments in terms of an induced dipole mechanism, using a sum
of three different polarizability terms to reproduce the saturation data.
Other electric birefringence, electric dichroism, conductivity dispersion,
and dielectric studies have been interpreted in terms of an induced dipole
mechanism,'-'l a permanent dipole m e ~ h a n i s r n , l ~usually - ~ ~ attributed
to a slow counterion fluctuation along the long axis of the DNA mole-
cu1e13J7J9-25or to a saturation of the induced dipole in high electric
or to a combination of permanent and induced dipole
Still other electric field light ~ c a t t e r i n gelectric
,~~
b i r e f r i n g e n ~ e , ~ ~and
- 3 ~d i e l e ~ t r i c ' ~studies
J ~ ~ ~ have
~ been interpreted in
terms of a transverse dipole moment or a slow counterion fluctuation across
the transverse axis of the DNA molecule.
The results of many of these studies have been complicated by the
polydispersity of the samples (frequently calf thymus DNA and/or soni-
cated fragments thereof) and by uncertainties in the molecular weight. In
addition, in many cases the DNA concentration was not low enough to
prevent intermolecular interaction^.^^ Since it is now possible, with the
aid of recombinant DNA technology (for reviews, see Refs. 39 and 40) and
site-specific endonucleases, to obtain DNA fragments of known size and
fully duplex character (for a more detailed discussion, see Ref. 41), the
questions of the Kerr law behavior of DNA and the mechanism of orien-
tation in an electric field can be investigated with monodisperse DNA
fragments of known molecular weight.
In this communication, birefringence saturation curves have been de-
termined for DNA fragments ranging in size from 80 to 4364 base pairs (bp;
5.7 X lo3to 2.9 X lo6 daltons). All fragments obeyed the Kerr law at suf-
ficiently low field strengths. The optical anisotropy factor, a measure of
the molecular asymmetry, is constant over this molecular weight range.
The rise of the birefringence of fragments I350 bp is symmetric with the
decay a t low field strengths, indicating orientation by an induced dipole
mechanism, The electric polarizability is proportional to the square of
molecular length. Comparisons are made with other values in the literature
and with available theories.

EXPERIMENTAL
Basic Theory
When an electric field is applied to a solution of polar macromolecules,
the macromolecules tend to orient in the field. If plane-polarized light,
with its plane of polarization rotated 45O from the direction of the applied
field, is passed through the solution, the emergent beam will be elliptically
polarized, with a phase shift, or optical retardation 6 (in degrees), equal
to
 BIREFRINGENCE OF DNA FRAGMENTS 401

An = nli - nl = 6X/3601 (1)

where An is the birefringence, the difference in refractive index of the so-
lution parallel and perpendicular to the applied field, l is the path length
through the solution, and X is the wavelength of the incident light. At
sufficiently low field strengths, the birefringence obeys the Kerr l a ~ ~ 5 > ~ ~ :
An 6X
K,, = --
nC,,E2 360nlC,,E2
where K,, is the specific Kerr constant, n is the refractive index of the
solvent, C,, is the volume fraction of the solute molecules in the solution,
and E is the electric field strength. C,, is usually calculated from iTc, where
6 is the partial specific volume and c the concentration of the macromole-
cule in g/cm3.
For rigid symmetric particles which can be oriented by induced dipole
moments or by permanent dipole moments along the long axis, the specific
Kerr constant can also be e x p r e s ~ e d ~as~ , ~ ~ - ~ ~
Ks, = [ 2 d g l - g2)/n21@ (3)
where (gl - g2) is the optical anisotropy factor, the difference in optical
polarizability per unit volume of oriented particles, and @ is an orientation
function defined by43744

where 8 is the angle between the electric field E and the particle axis and
f ( 8 )is an angular orientation distribution function,

where U is the electrostatic energy

u = -PEB, cos 0 - 4 (all - a l ) ~ 2 cos2e (6)
and y is the apparent dipole moment along the long axis, B1 is an internal
field and (cull - a1) is the difference in induced polarizability
parallel and perpendicular to the long axis of the molecules.
+
Expansion of at low field strengths leads to35,42,43

where k is Boltzmann's constant and T the absolute temperature. For a

more convenient notation, we define
P =(yI31/kT)~ and Q = (all - al)/hT (8)
In conducting solutions, the electric polarizabilities involve the conduc-
tivities as well as the dielectric properties of the p a r t i c l e ~ : ~ ?as~ ~will be
discussed below.
402 STELLWAGEN

-
A t infinitely high electric field strengths, CP 1and the Kerr constant
becomes a function of the optical anisotropy factor only, regardless of the
mechanism of orientation of the particles in the electric field:
(&p)E-- = 27dg1 - g2)/n2 (9)
A suitable method of extrapolation to infinite field must be chosen.43
Peterlin and Stuart44have proposed a theoretical equation for the optical
anisotropy factor of ellipsoids with geometrical and optical axial sym-
metry:

(g1 - g2)
+
(nf - n:) [(nf - n2)(n; - n2)/n2](L2 - L1)
=
4& + +
2a[(nz - n2)/n211{1 2a[(n? - n2)/n2]1
(10)

where nl and n2 are the refractive indices of the particle parallel and per-
pendicular to the axis of symmetry, n is the refractive index of the solvent,
and L1 and L2 are elliptical integrals dependent only on the axial ratio of
the particles. For prolate ellipsoids of revolution with axial ratios 2 10,
Eq. (10) simplifies to

(g1 - g2) =
+
(nf - n;) [(n?- n q n ; - n2)/n2]27r
+
4 a { l [(n:- n2)/n2]2s)
(11)

The first term in Eq. (11)corresponds to the intrinsic anisotropy of the

particles, and the second term is a shape or form factor arising from the
difference in refractive index between the particle and the solvent.46 For
DNA, the intrinsic anisotropy is negative, because of the orientation of the
polarizable purine and pyrimidine bases perpendicular to the long axis of
the molecule. The form factor is always positive.46
The optical factor is related to the optical anisotropy/base pair, ( 7 1 -
~ 2 1 ,

(71- 7 2 ) = (g1 - g2)V (12)

where V is the volume of the particle (V = i7M/Na),with M the molecular
weight and N a being Avogadro's number.
The decay of the birefringence of a monodisperse solution of rigid axially
symmetric molecules is given by47848
An/(An)+o = e - t / T (13)
where An is the birefringence at any time t, (An)t=ois the birefringence
when the electric field is removed, and 7,the relaxation time, is related to
0, the rotational diffusion constant, by
8 = 1/67 (14)
The rotational diffusion constant can be related to particle length by an
equation given by B r ~ e r s r n a ~ ~

3kT [In 2p - 1.57 + 7

0=-
87r77a3
(In
-
2p
- 0.28)2]
 BIREFRINGENCE OF DNA FRAGMENTS 403

where q is the solvent viscosity, p is the axial ratio, and a is one-half the
length of the particle.
The rise of the birefringence in a monodisperse solution of rigid axially
symmetric particles a t low field strength is given by35147

If the particles have no dipole moment, P = 0,

AnR/Ano = 1 - e-60t
and the rise of the birefringence is symmetric with the decay.
More extensive discussions of the theory of electric birefringence are
given in two recent reviews.35.42

Apparatus and Procedures

The birefringence apparatus used in this study is described in detail in
a companion paper.41 The light source was a model S-101 (CW-Radiation
Inc.) 0.5 mW He-Ne laser with an emission wavelength of 633 nm, polarized
a t 45" with respect to the laser base. The Kerr cell was a 1.00-cm path-
length glass spectrophotometer cell chosen for its negligible strain bire-
fringence. The platinum electrodes were mounted on a delran electrode
support similar in design to that used by other^.^^,^^ The electrode sepa-
ration was 2.2 mm; the cell could be filled with 0.6 cm3 solution. The cell
was placed in a water-jacketed cell holder cooled with running tap water.
Temperatures of different birefringence experiments ranged from 16 to
21"C; all birefringence relaxation times were corrected to a standard
temperature of 2OoC by the following relation:
720 = (Tobd293)7obs
(7?20/7?T) (18)
The output from the birefringence cell, after passing through a quar-
ter-wave plate and an analyzer typically rotated loo from the crossed po-
sition, was detected by a PIN-5 photodiode (United Detector Technology)
and LM308 optical amplifier (National Semiconductor). The overall time
constant of the photodetection system was ca. 1 psec, determined with
propylene carbonate or water in the birefringence cell. The stray-light
constant of the birefringence apparatus was 1.1 X
The birefringence signals were recorded on a Nicolet model 1090 digital
oscilloscope equipped with a model 92 dual-channel differential amplifier.
The data were stored and analyzed on an Altos/8000 minicomputer
equipped with SOROC I&-120 keyboard and display terminal; the software
for this system was designed and constructed by Sorrento Valley Associates,
Sorrento Valley, California. Data could be transferred directly from the
computer to the oscilloscope and vice versa.
The pulse generator and amplifier produced square-wave pulses which
could be varied from 1 psec to 1 msec in duration and 500 V/cm to 25 kV/cm
404 STELLWAGEN

in amplitude. The pulse generator was phase locked with the laser beam
to allow multiple pulsing an$ averaging of the birefringence signals. The
exact voltage of the high-voltage pulse was measured by means of a 1OOO:l
stepdown probe (Tektronix P6013A). The rise time of the pulse was less
than 1 psec. The decay time varied somewhat with the conductivity of the
solution in the cell; with a 5 Kfl resistor in place of the cell, the time constant
for the decay of the pulse was 1.7 psec.
The birefringence of the various solutions was calculated from the exact
equation for the optical s y ~ t e r n ~ ~ , ~ ~ :
&/Ia = [sin2(a + 6/2) - sin2a]/sinZa (19)
where a is the angle through which the analyzer is rotated from the crossed
position, AI is the change in light intensity caused by application of the
field, I , is the light intensity transmitted by the analyzer set a degrees from
the crossed position in the absence of the field, and 6 is the retardation. To
analyze the transient behavior of the birefringence, Eq. (19) was approxi-
mated by
6 = (A1/Ia)a (20)
with a resultant error in 6 of less than 2%.
To measure the birefringence relaxation times, the DNA solutions were
pulsed 4-8 times, reversing the polarity of the pulse each time the solution
was pulsed. The resulting birefringence signals were stored and averaged,
and the corresponding base lines were subtracted by the computer.41
With DNA fragments larger than 380 bp it was necessary to use very short
orienting pulses, on the order of 8 psec, in order to get accurate values of
q o n g 4 1 Longer orienting pulses at a given E resulted in a decrease of the
absolute, as well as the relative, amplitude of qong, as shown in Table I for

TABLE I
Dependence of Apparent Relaxation Time on Pulse Length
Pulse Length, qong Absolute Amplitude Relative
Sample Buffer (psec) (psec) (arbitraryunits) Amplitude
Y5 TE2 5 170 0.20 0.34
(1080 bp 8 175 0.20 0.31
7.6 pglml, 10 165 0.17 0.26
E = 10.9 kV/ 15 170 0.13 0.18
cm) 308 42a 0.52a 0.908
TElO 5 150 0.18 0.32
8 150 0.18 0.31
10 110 0.15 0.25
Y4 TE2 8 136 0.19 0.32
(940 bp 40 48 0.41 0.90
7.7 pglml, 608 358 0.44a O.8Oa
E = 3.4 kV/ 80 26 0.45 0.90
cm)
a Steady-state orienting pulse.
 BIREFRINGENCE OF DNA FRAGMENTS 405

Y5, 1080 bp. The pulse length a t which the amplitude of qong began to
decrease was less than half of the pulse length needed to reach steady-state
orientation. Longer orienting pulses could be used in TE2 buffer than in
TElO buffer before 7long began to decrease. Increases in pulse length be-
yond that required to reach steady-state orientation caused a further de-
crease in the apparent value of the relaxation time, as shown in Table I1
for Y4,940 bp. The relaxation times of DNA fragments smaller than 380
bp were unaffected by the length of the orienting pulse.
Two explanations may be suggested for the decrease of 71ong with in-
creasing pulse length. (1)The longer DNA molecules may be folded or bent
into more compact configurations by electric fields applied for a relatively
long duration. Since the specific Kerr constant is larger for more highly
extended molecules (see below), folding or bending would result in a de-
crease of the absolute amplitude of the birefringent component associated
with qongRelatively short orienting pulses would have caused a relatively
small perturbation in the average conformation of the population of DNA
molecules, and therefore would not have affected the amplitude of 710ng.
(2) Long pulses may promote aggregation of the larger DNA fragments.
Preliminary experiments with relatively concentrated solutions of sample
B11 (ca. 620 bp) in TElO buffer (DNA concentration I14 pg/ml) suggested
that pulses longer than required to reach steady-state orientation in electric
fields I10 kV/cm caused a small positively birefringent component with
a very long time constant (ca. 100 ysec) to appear in the decay of the bire-
fringence. The amplitude of this positive birefringence increased with
increasing pulse length, accompanied by a corresponding decrease in the
absolute amplitude and apparent time constant of the steady-state (neg-
ative) birefringence. The absolute amplitude of the positive birefringence
was typically about 3 4 %that of the steady-state (negative) birefringence.
The positive birefringence could be eliminated completely, even in con-
centrated solutions, by shortening the pulse length to values equal to or
less than required to reach steady-state orientation. Positive birefringence
was not observed for any of the other DNA fragments.
T o measure the birefringence saturation curves, pulses were applied to
the solutions which were long enough to reach steady-state orientation a t
each different field strength; these pulses ranged from 20 to 200 ysec in
duration, depending on the amplitude of the applied electric field and the
size of the DNA fragment. Starting a t a moderate value of E, the electric
field was decreased stepwise to the lowest values at which a signal could
be detected (with signal averaging), then increased stepwise to reach es-
sentially complete orientation, and then decreased again to moderate
values. The retardations measured at moderate E after high-voltage pulses
(ca. 20 kV/cm) had been applied to the solution were always the same as
those originally measured. Allowing the solution to stand in the cell 1-2
hr in the middle of a saturation study also did not affect the values of the
retardation observed. The anomalous high-field effect observed by
O’Konski and Stellwagensl and by Pollack and G l i ~ was k ~ not
~ observed
406 STELLWAGEN

for any of the DNA fragments studied here. The peculiar birefringence
signals observed by Colson et al.53in concentrated solutions of sonicated
DNA fragments were also not observed here.
Saturation curves were repeated 2-6 times for each of the DNA frag-
ments, using fresh solutions for each saturation curve. Parameters given
in the tables and figures are averaged values, unless otherwise indicated.
The observed birefringence was corrected if necessary for the birefringence
of the solvent. This correction became important only a t E 1 10 kV/cm,
where the birefringence of the DNA fragments began to approach satura-
tion. The Kerr constant of water was found to be 1.71 X cgs esu (1.90
X V-2 m) a t the wavelength used here (633 nm).
Optical density melting curves were measured with a Gilford model 2000
recording spectrophotometer containing a calibrated thermistor in an
adjacent cell. The temperature was increased a t a rate of 0.66"C/min.
DNA concentrations were determined using a value of A (260 nm):50 pg
DNA/ml = 1.0 (E. J. Gubbins et al., unpublished; Ref. 54).
A birefringence melting curve was measured for one of the fragments
using a Laude K-2/RD (Brinkman Instruments) constant-temperature bath
to control the temperature of the birefringence cell. The temperature was
raised stepwise, waiting a t least 5 min after each change of temperature
before measuring the birefringence. Samples were changed in the middle
of the birefringence curve with no detectable difference in either the
steady-state birefringence signal or the relaxation time.

DNA Fragments

Plasmid 826B"
Escherichia coli cells containing plasmid pMB9 were grown a t 37°C in
M9 medium54containing added MgC12, glucose, thiamine, MgS04, glucose
thiamine, CaC12, Casamino acids, and tetracycline. Growth was followed
until OD = 0.5-0.6, followed by chloramphenicol amplification (170 mg
chloramphenicolfl. culture) overnight.54 The harvested cells were lysed
with lysozyme,55the cell debris spun down, and the DNA banded in a ce-
sium chloride-ethidium bromide gradient (refractive index = 1.3965; 10
mg ethidium bromide/ml DNA solution) a t 39,500 rpm for 2 days in a
fixed-angle 60-Ti rotor in a Beckman L3-40 centrifuge. The DNA was then
visualized with long-wavelength uv light and the superhelical plasmid DNA
removed with a spinal tap needle inserted from the top. The plasmid-
containing fraction was extracted three times with n-butanol, to remove
ethidium bromide, and passed through a 2 X 20-cm Biogel A15 column,
eluting with a solution containing 10 mM Tris, pH 8.5,20 mM EDTA, and
0.5M NaC1. The plasmid eluted in the void volume in fractions 7-9. These
fractions were pooled, the plasmid precipitated with 2.5 volumes cold
ethanol, redissolved in TElO buffer (10 mM Tris, pH 8.0, 1 mM EDTA)
* Gubbins et al., unpublished.
 BIREFRINGENCE OF DNA FRAGMENTS 407

and stored at -20°C. The yield was about 1g plasmid per liter of culture.
All work involving this two-host plasmid system was performed in accor-
dance with current NIH guidelines for recombinant DNA research.

Restriction Fragments
Restriction fragments were prepared by incubating 1-mg portions of
+
plasmid 826B with Hind I1 I11 (in 6 mM NaC1,7 mM MgC12,7 mM Tris,
pH 7.4) or with Alu I (in 60 mM Tris, pH 7.4, 60 mM MgC12, 100 pg/ml
gelatin, and 4 pl/ml 0-mercaptoethanol) for 24-36 hr a t 37°C. After the
reaction was complete, 1/10 volume of a solution containing 50%sucrose,
2 X TBE buffer (5 m M Tris borate, pH 8.3,l mM EDTA) and 0.4%brom-
phenol blue (tracking dye) was added to the DNA solution and the mixture
applied to the single well of a 17 X 20 X 0.32-cm, 6% acrylamide slab gel
(monomer:bis ratio, 29:l). The mixture was electrophoresed a t 350 V (48
mA) for 14-2 hr, using TBE as the running buffer. After electrophoresis,
the gel was stained lightly with ethidium bromide, visualized with long-
wavelength uv light, and the bands containing the various fragments cut
out with a scalpel. The gel slices were mashed with a glass rod or, in some
preparations, forced through a large-bore syringe, incubated overnight with
shaking in enough 20 mM Tris buffer to make a slurry, and centrifuged.
The supernatants were removed from the gel, filtered through glass wool,
and the DNA concentrated on small DEAE cellulose columns (Whatman
DE52) with 34-mm gel beds. The fragments were eluted from the columns
with a solution of 0.5M Tris buffer and 0.5M sodium acetate, precipitated
with 2 volumes cold ethanol, and redissolved in 100 p1 TElO buffer. The
fragments were stored in TElO buffer at -2O"C, and were stable for more
than a year.
Molecular weights of about half the restriction fragments were deter-
mined from the nucleotide sequence (J.Hartley, personal communication)
and are known exactly. Molecular weights of the other fragments were
estimated from electrophoresis on a 17 X 30 X 0.16-cm, 4.5% acrylamide
slab gel (monomer:bis = 29:1), using TBE as the running buffer at 350 V
for 1hr. Thirteen sequenced fragments of 6x174 cut with Hind I1 I11 +
(kindly provided by E. Gubbins), spanning the molecular weight range of
interest, were run simultaneously. Some of the larger fragments were also
sized on 2% agarose gels by P. Hagerman (personal communication).
To increase the yield of some of the smaller fragments, several fragments
close in molecular weight were combined into one sample, as shown in Fig.
1,which is a photograph of the analytical gel of the Alu I restriction frag-
ments. Figure 1 also shows that no sample contained DNA fragments
appreciably larger or smaller than the given molecular weight. A summary
of the samples used in this study, the molecular weights of their constituent
fragments, and average molecular weights are given in Table 11. The
4364-bp sample was prepared from plasmid ~ B R 3 2 2 ~ byb a single cut with
EcoRI,~ and
~ was kindly supplied by Hagerman.41 The sample called G2,
408 STELLWAGEN

Fig. 1. Photograph of analytical 4.5%acrylamide slab gel (monomer:bis= 2 9 1 ) containing

fragments produced by an Alu I digestion of plasmid 826B, stained with ethidium bromide.
The running buffer was TBE buffer at 350 V for 1 hr. From left to right, the samples and
their designations are: B12, B11 [used in Fig. 3(b)], B10, B9, B8, B7, standards (6x174 cut
+
with Hind I1 III), B6, B5, B4, B3, B2, B1.

Y2 was actually two different preparations of the same fragment, measured

separately, and the results (which were the same within experimental error)
averaged.

Buffer
The birefringence of most samples was measured either in TE2 buffer
(0.2 mM Tris, pH 8.0,0.02 mM EDTA) or in TElO buffer (10 mM Tris, pH
8.0, 0.1 mM EDTA), diluting the DNA stock solutions with distilled
deionized water or TElO buffer to the desired DNA concentration. Tris
buffers were chosen because of their low conductivities, making it possible
to extend the birefringence measurements to higher electric fields. In
addition, DNA is stable at lower ionic strengths if the pH of the solvent is
greater than about
 TABLE I1
Molecular Weights, Relaxation Times, and Calculated Lengths of Various DNA Fragments in TE2 Buffer
Sample Base Pairs Average Base Pairs Tiunga (wet) Relative Amplitude a t High E Lengthb (A)
N
B2 79,80 80 - - -
B3 92,104 98 - - -
4
E
B4 125,127,136 129 1.9 1.00 440 z
B5 151,166,166 161 2.9 1.00 520
B6 200,221,231 217 6.2 1.00 700
B7 257 257 10.0 0.87 850 2M
B8 281,284,306,307 294 13.5 0.79 950
B9 374,381,386,402,403 389 21.3 0.61 1130
B10 45O,48Oc 465 40 0.54 1430
G2, Y2 623,633 628 60 0.35 1660 U
G3 782 782 90 0.30 1930 z
B12 83OC 830 99 0.24 2000
Y4 940d 940 130 0.22 2220
Y5 1080d 1080 190 0.25 2540 !i
Y6 1314 1314 280 0.33 2900 E
PBR322 4364 4364 - - -

a All measurements corrected to 20°C.

Calculated from Broersma’s equation.
Determined by acrylamide electrophoresis (see text).
Determined by acrylamide and agarose electrophoresis (see text).

Ip
0
(0
410 STELLWAGEN

Melting Curves

Melting curves were measured for three of the fragments in TE2 buffer
and are shown in Fig. 2. Obvious differences are observed in the melting
temperatures of the different fragments, possibly due to differences in base
sequence and composition or to differences in DNA concentration.
However, in all three cases the DNA fragments retained their native
structure at temperatures well above the temperature used for the bire-
fringence experiments (2OOC).

RESULTS AND DISCUSSION

Concentration Dependence
The specific birefringence ( 6 / c )of the various DNA restriction fragments
was independent of concentration below about 16-18 pg/ml, as shown in
Fig. 3(a) for samples B6,217 bp, and B9, 389 bp. The saturation curves
of the two samples converged toward the same value of 6/c at high values
of E, but differed at low E. The limiting slope of the low-field portions of
the two curves was 2.0, showing that the Kerr la^^^,^^ was obeyed at low
fields. A t concentrations greater than about 16-18 pg/ml, the specific
birefringence of the DNA fragments decreased, as shown in Fig. 3(b) for
sample B11. Therefore, all measurements reported here were obtained
at DNA concentrations I18 pg/ml.

Buffer and Buffer Concentration

The specific birefringence of the different DNA fragments depended on
the concentration and type of added low-molecular-weight salt, as shown
in Fig. 4(a) for sample B5, 161 bp. The decrease in the specific birefrin-

I r I
IOC

x
Change

0
I I I
so" 5on 6(
Temp.
Fig. 2. Optical density melting curves of three representative fragments in TE2 buffer,
measured at 260 nm: ( A ) B6,217 bp, 15.4 pg/ml; ( 0 )B10,465 bp, 4.7 pg/ml; (0)
Y4,940 bp,
6.6 pg/ml.
 BIREFRINGENCE OF DNA FRAGMENTS 411

1.c I I 1

a b

0.1

- blC .
deg/vgfml

1 10 1 10
E, hvlcm E. kdcm

Fig. 3. (a) Saturation curves of samples B6,217 bp, and B9,380 bp, a t different concen-
trations in TE2 buffer: (A)B6, 8.9 pglml; ( A ) B6, 16.4 pg/ml; ( 0 )B9, 6.8 hg/ml; (O), B9,
16.5 pg/ml. (b) Saturation curves of sample B11,620 bp, in TElO buffer, as a function of DNA
concentration: (v)3.5 pg/ml; ( A ) 7.0 pg/ml; ( 0 )14 pg/ml; (n)28 pg/ml.

gence with increasing buffer concentration was more pronounced for the
higher-molecular-weight samples, especially in the region of intermediate
E above the Kerr law region, as shown in Fig. 4(b) for sample Y6,1314 bp.
r l

0.1 -
-be,

deg/vg /ml

0.01 -

1 10 1 10
E, kvlcm E, hvlcm

Fig. 4. Saturation curves of DNA fragments in solutions containing different added salts.
(a) B5,161 bp, in ( 0 )TE2 buffer; (A)TE2 buffer plus 1mM NaC1; (0) TE2 buffer plus 1mM
MgC12. (b) Y6,1314 bp, in (A)TE1 buffer; (0) TE2 buffer; (0)TElO buffer; (v)TE1 buffer
plus 1mM NaCl; ( O ) ,TE1 buffer plus 1mM MgC12.
412 STELLWAGEN

The saturation curves were displaced to higher fields with increasing solvent
conductivity, probably due to the fact that in conducting solutions, the
polarizabilities are a function of the conductivities, as well as of the di-
electric constants of the medium.42,45359
A theory of the ionic polarization of polyelectrolytes, based on the motion
of counterions along the surface of the polyion, has been developed by
O’Konski and K r a ~ s e . ~ For ~ , rodlike
~ ~ l ~ particles
~ of high axial ratio, the
surface conductivity u of counterions bound to the surface but mobile in
the longitudinal direction was shown to be equivalent to an anisometric
volume conductivity:
K, = 2u/b, Kb = a/b (21)
where K, and Kb are the equivalent volume conductivities along the sym-
metry and transverse axes, respectively, a is one-half the length of the
cylinder, and b is the radius. The surface conductivity can be calculated
from the charge density per unit surface, which for cylinders is
(T = ueznl4aab (22)
where u is the mobility of the counterions, usually assumed equal to the
mobility in the bulk of the solution as a first approximation, n is the total
number of counterions of valence z , e is the electronic charge, and a is
one-half the length of the cylinder. For a molecule such as DNA, where
the high charge density results in a constant fraction of the counterions
being condensed on the molecule,6M2it would seem appropriate to multiply
n by 6,the fraction of condensed counterions. Thus,
u = &.~e(bp)/2nab (23)
since nz = 2 X bp.
The equation of O’Konski and Krause for the Kerr constant of a highly
conducting rod-like particle with no permanent dipole moment, immersed
in a medium of much lower conductivity, can be simplified to59:

where V is the volume of the particle, 6 is the dielectric constant of the

solvent, KO is the conductivity of the solvent, n is the refractive index of the
solution, and the A, are depolarization If the volume of the DNA
fragment is taken as the unhydrated volume determined from the number
of base pairs multiplied by 3.4 A, the average extensionlbase pair63and a
diameter of 26 A,64 and the fraction of condensed counterions is taken to
be 0.76 for monovalent cations60-62and 0.88 for divalent cations,61,62the
dependence of the Kerr constant on solvent conductivity can be calculated
from Eq. (24). These theoretical values are compared with the experi-
mental values in Table 111. Although there is a difference between the
calculated and observed values of Ksp,especially for the two larger samples,
both the calculated and observed values decrease with increasing solvent
 BIREFRINGENCE OF DNA FRAGMENTS 413

TABLE I11
Dependence of Specific Kerr Constant on Solvent Conductivity
K,,, x 105
SamDle Kn. x 104 Calcd. EXD. Calcd.a
B5 0.082 4.2 2.4 4.2
(161 bp) 0.104 4.2 3.3 4.2
1.22 3.3 2.4 3.3
2.60b 1.8 1.1 1.8

B8 0.067 24 13 9.4
(294 bp) 0.104 24 12 9.4
1.22 15 7 7.3
2.60b 9.7 1.7 3.9

Y6 0.067 940 44 40
(1314 bp) 0.104 809 55 40
0.41 332 32 39
1.22 131 31 32
2.60b 65 4.8 17
* Calculated using the depolarizing factor of sample B5.
1 mM MgC12.

conductivity. The agreement between the calculated and observed values

of K,, could be improved by choosing a larger effective diameter for the
larger fragments, which would allow for more bending and coiling of these
molecules. However, agreement is also obtained by assuming that the
factor in brackets in Eq. (24), [l/A, +
K O / K , ] , is constant for all the frag-
ments; i.e., depolarization takes place only over a distance of the order of
one persistence length. Assuming that the factor in brackets calculated
for B5, 161 bp, is representative of the depolarization factors of all frag-
ments that size and larger, the values of K,, in the last column of Table I11
are calculated from Eq. (24). These calculated values are of the same order
of magnitude as the observed values and decrease with increasing solvent
conductivity, as observed experimentally. Specific Kerr constants of all
the fragments in TE2 buffer, calculated from Eq. (24) and the depolariza-
tion factor of B5, are given in the fifth column of Table IV.
An alternative formulation of the ionic-strength dependence of the in-
duced polarizability, given by Manning,65 suggests that the polarizability,
and hence the specific Kerr constant, increases with increasing ionic
strength, in contrast to the experimental results.
The specific Kerr constants of all three DNA fragments in the solution
of highest conductivity, 1 mM MgC12, were much lower than observed in
1mM NaCl or in TElO buffer (1mM Tris). Similar results have been re-
ported by Rau and Bloomfield (1) using mixed Na+-Mg2+ solutions a t
constant ionic strength. However, previous experiments using calf thymus
DNA36 showed that the specific birefringence of DNA in a dilute MgClz
solution (Mg2+/P = 1:l)was essentially equal to that observed in Tris buffer
solutions of the same conductivity, while the specific birefringence observed
414 STELLWAGEN

TABLE IV
Electrical Parameters of DNA Fragments in TE2 Buffer
-K,,, x 105
-(6/cE%-o (cgs esu) (ff!!-al) cc-
Sample Base Pair (denlfiglrnl) Exp. Calcd. (X 10l6cm3) (D)
B2 80 0.48 1.1a 0.71 0.82a
B3 98 0.66 1.6 1.4 1.3
B4 129 0.98 2.3 2.6 2.0
B5 161 1.4 3.3 4.2 3.0 -
B6 217 3.2 7.6 6.5 4.8 6200
B7 257 4.5 10.7 7.6 7.3 6200
B8 294 4.8 11.5 8.7 8.8 6200
B9 389 5.6 13 12 8.9 5900
B10 465 7.6 18 14 12 6300
G2,Y2 628 4.0 9.5 19 8.1 6400
G3 782 6.4 15 23 11.6 6900
B12 830 7.2 17 24 13 6100
Y4 940 8.4 20 28 15 6700
Y5 1080 12 28 32 21 6000
Y6 1314 23 55 40 38 7000
PBR322 4364 30 71 130 50 6800
a Determined in T E 8 buffer (8 mM Tris, 0.8 mM EDTA).

in a solution of higher Mg2+concentration (Mg2+/P= 1O:l) was only about

half as large as observed in Tris andlor cacodylate buffer solutions of
comparable conductivities. The Mg2+/Pratio in the 1mMMgClz solutions
used here and in the Rau-Bloomfield experiments were both >> 1,
suggesting that the Mg2+-DNA interaction needs to be better characterized
before drawing conclusions about the effect of Mg2+on the electric polar-
izability of DNA. In no case has an increase in the specific Kerr constant
been observed in the presence of Mg2+, as would be expected from the
Mandel equation for electric polarizability, Eq. (25).

Temperature Dependence
The birefringence of one of the DNA samples, B6,217 bp, was determined
as a function of temperature at 8.25 kVIcm, using steady-state orienting
pulses. The results are shown in Fig. 5, where the change in optical density
is plotted as 100% - (% change) to facilitate comparison with the bire-
fringence results. The birefringence relaxation times have been corrected
to 20°C (temperature and viscosity corrections) by means of Eq. (16). The
corrected relaxation times remained approximately constant up to and
through the transition region, indicating that the molecules remained highly
extended in this region. After the transition, the corrected relaxation times
appeared to increase slightly, although the accuracy of these data is poor
because of the marked decrease in signal amplitude and the large correc-
tions required to standardize the data a t 20°C. The birefringence decay
curves were unimodal a t all temperatures.
 BIREFRINGENCE OF DNA FRAGMENTS 415

I I U I

101

%
Change

5(

I I .. I
20" 40" 60"
Temp

Fig. 5. Comparison of the melting curve of optical density (v),plotted as loo%-% change,
with the melting curve of the steady-state birefringence (0) and field-free decay of the bire-
fringence ( 0 )of fragment B6,217 bp, in TE2 buffer. E = 8.25 kV/cm; DNA concentration,
17.5 pg/ml. Samples were changed in the middle of the birefringence curve with no detectable
difference in either the steady-state signals or the birefringence relaxation times. The re-
laxation times have been corrected to 20°C by Eq. (16);the amplitude of the birefringence
has been corrected to 20°C assuming a 1/T2 dependence on temperature (see text). The bi-
refringence signal is small and positive between 58-65OC.

The amplitude of the birefringence was corrected to 20°C by assuming

that the birefringence was proportional to 1/T2;assuming the birefringence
to be proportional to 1/T did not result in a constant value of the birefrin-
gence at temperatures below the transition region. With a 1JT2correction
for the change in amplitude of the birefringence with increasing tempera-
ture, it can be seen that the "melting curve" for electric birefringence follows
the fine structure of the melting curve of optical density, except that the
first half of the birefringence curve occurs at temperatures about 6°C lower
than that of the optical density curve. According to Manning,6othe de-
crease of the melting temperature can be attributed to the increased free
energy of condensed counterions in an electric field. Helical DNA, with
its greater charge density, binds a greater number of counterions than coil
DNA and therefore has a greater increase in free energy in an electric field
than coil DNA, leading to strand separation a t a lower temperature. The
decrease in amplitude of the birefringence curve in the transition region,
like that of optical density, must be due to progressive disorientation of the
bases with increasing temperature.
Manning60.'j6has calculated the work required to remove a charge per-
pendicular to the long axis of an unoriented polyion in an electric field. For
416 STELLWAGEN

a solution of very low ionic strength, the melting temperature is predicted

to decrease about 0.5"C in a field of 8 kV/cm, much less than the observed
decrease. The discrepancy may be due to the neglect of orientation effects
in the theory.
At temperatures above the transition region, the birefringence appeared
to become slightly positive, in agreement with previous observation^.^^-^^
However, the amplitude of the positive birefringence was only about 1%
that of the original negative birefringence.

Decay of the Birefringence

The decay of the birefringence of DNA fragments 5 217 bp was unimodal
a t all electric field strengths. A typical example is shown in Fig. 6 for B6,
217 bp. DNA fragments between 257 and 465 bp exhibited a unimodal
birefringence decay at low and moderate electric fields, but at high electric
fields the decay of the birefringence also contained a faster component.
The relative amplitude of the component with the slower relaxation time
(here called qong) decreased with increasingE for these fragments, as shown
in Fig. 7(a), although the values of 710ng were independent of E (Fig. 8).
DNA fragments larger than 465 bp did not exhibit a unimodal birefringence
decay even a t the lowest field strengths at which the relaxation times could
be measured accurately.
As the electric field strength was increased into the saturation region,
the relative amplitude of qong appeared to level off and approach a constant

I0 I I
20
I
30
lo Nsec.
Fig. 6. Semilog plot of the birefringence decay of sample B6,217 bp, in TElO buffer. DNA
concentration, 12.8 pg/ml; E = 8 kV/cm. The decay curve represents an average of 8 indi-
vidual curves. T = 5.9 usec.
 BIREFRINGENCE OF DNA FRAGMENTS 417

1.0

Relative
Amplitude

0.5

I I I ' I

(b)
Fig. 7(a) Dependence of the relative amplitude of TIong on electric field strength for several
DNA fragments in TE2 buffer. The curves for fragments 5 465 bp include data obtained
from both 8 gsec and steady-state orienting pulses. (b) Dependence of the relative amplitude
of ~l~~~ in saturating fields on the number of base pairs in the fragment. The DNA concen-
tration in all cases was ca. 10 gg/ml in TE2 buffer.

value. The relative amplitude of qong at high E was roughly inversely

proportional to molecular weight, as shown in Fig. 7(b), for fragments I
250 bp.
The values of the long relaxation times for the different DNA fragments
418 STELLWAGEN

r I I 1

300 - Q
-

100
-

rlong '
pec.
5

30 -

c;) 6) 294

10 8-a IJ I

E , kvlcm
Fig. 8. Birefringence relaxation times (qong) obtained with 8-wsec orienting pulses plotted
as a function of electric field strength. The DNA concentration ranged from 5.7 to 12.4 gg/ml
for the different samples, except (O), 19 pg/ml. The buffers used were (0) TE2 buffer, and
(A),(A),and (9)TElO buffer.

were essentially independent of buffer concentration in TE2 and TElO

buffer and independent of electric field strength and DNA concentration,
as shown in Fig. 8. Hence these relaxation times are characteristic of the
individual fragments and are not influenced by experimental conditions.
The values of qong observed for each of the DNA fragments are given in
Table 11; these values are averages of 6-30 separate determinations for each
sample. The relative amplitudes of qong a t high E are also given in Table
11.
Lengths were calculated for the various DNA fragments from the values
and the Broersma equation, Eq. (15), using a molecular diameter
of 7
Of 26 . These lengths are tabulated in Table 11, and are plotted in Fig.
9 as a function of the number of base pairs in the samples. The drawn line
corresponds to 3.4 A h p . Coiling becomes important for DNA fragments
larger than 300 bp, as evidenced by the increasing deviation of these points
from the drawn line; this topic will be discussed ~ e p a r a t e l y . ~ ~
The short relaxation times observed in the decay of the birefringence
of the larger DNA fragments may be associated with bending or twisting
motions of the DNA molecules,7l or with motions associated with the return
of the DNA molecules to their equilibrium configurations after having been
perturbed by the field and will not be discussed further.
Rise of the Birefringence
At very low field strengths the rise of the birefringence was accurately
symmetrical with the decay, as shown in Fig. 10 for three of the DNA
fragments. Therefore, the DNA fragments orient in an electric field by
an induced dipole m e ~ h a n i s r n . ~ ~ , ~ ~ . ~ ~
 BIREFRINGENCE OF DNA FRAGMENTS 419

3000 1 I I ' 0

Base Pairs
Fig. 9. Dependence of the length calculated from the Broersma equation and qong
on the
number of base pairs in the fragment. The drawn line corresponds to 3.4 h p .
At higher electric fields the rise of the birefringence became faster than
the decay because of the additional orienting torque exerted by the field.42,43
The threshold field strength at which the rise of the birefringence became
faster than the decay, E t h , was approximately equal to the field strength
above which Kerr law behavior was no longer observed, as shown in Fig.
11. Hence, rise times (such as obtained from a temperature-jump appa-
ratus) can only be used to characterize molecular reorientation within the
Kerr law region. In addition, the rise of the birefringence of large flexible
DNA molecules may be faster than the decay even within the Kerr law re-
gi0n.l

Birefringence in Saturating Fields and t h e Optical Factor o f DNA

The specific birefringence of all the DNA fragments tended toward the
same limit at high values of E, as can be seen from Figs. 3 and 4. For all
fragments except the smallest (B2,80 bp), sufficientiy high electric fields
were reached that the specific birefringence, corrected for the birefringence
of the solvent, appeared to reach a constant, or nearly constant, value of
6/c. These limiting values of 6/c are tabulated in Table V; each value
represents an average of 2-7 separate determinations for each sample. The
average value of 6,/c for all fragments was -0.31 f 0.02 deglpglml. The
high-field data were also extrapolated to infinite field strength by plotting
6/c vs 1/E and extrapolating to 1/E = 0. A typical extrapolation is shown
in Fig. 12. The extrapolated values of 6Jc, again averages of 2-7 separate
determinations for each sample, are given in Table V; the average for all
fragments was -0.38 f 0.02 deg/pg/ml. Extrapolation of the high-field
data to infinite field strength by plotting 6/c vs 1/E2and extrapolating to
1/E2= 0 resulted in an average value of -0.33 f 0.02, very close to the ex-
perimentally observed average value in high fields.
420 STELLWAGEN

I I
0 5 10 15
psec.

0.01I I I I
0 20 40
psec.
Fig. 10. Comparison of the rise and decay of the birefringence for three DNA fragments:
(O),decay, 6/60; ( 0 )rise (1- 6/60). (a) B4,129 bp, 5.5 pg/ml in TElO buffer, E = 10 kV/cm
and B6, 217 bp, 16.4 pg/ml in TE2 buffer, E = 4.5 kV/cm; (b) B8,294 bp, 9.2 pg/ml in TE1
buffer, E = 2.1 kV/cm.

Using the values of 6,Ic obtained from the 1IE extrapolation of the bi-
refringence of each DNA fragment, optical factors (gl - gz) were calculated
for each fragment from Eqs. (1)and (8),taking the partial specific volume
of DNA to be 0.50 cm3/g73,74in the dilute TE2 buffers used here. These
optical factors are given in column 6 of Table V. The average value of the
optical factor for all fragments was -0.028 f 0.002. This directly deter-
mined value may be compared with the value of -0.0215 obtained by
Sokerov and Weill (2) from a comparison of the electric birefringence and
 BIREFRINGENCE OF DNA FRAGMENTS 421

(EL P . ) ~ ' lo3

Fig. 11. Dependence of Ethr the field strength a t which the rise of the birefringence became
faster than the decay (A)and the field strength above which the Kerr law was no longer obeyed
(0) on the number of base pairs in the DNA fragments.

electric dichroism of sonicated fragments of calf thymus DNA complexed

with the dye acridine orange. Emonds-Alt et al.* obtained a value of
-0.0205 f 0.0003 for the optical factor of DNA by analyzing the shape of
the electric birefringence saturation curve, assuming a pure induced dipole
orientation mechanism with three different polarizabilities. The corre-
sponding value in the present study would be calculated from a 1lE ex-
trapolation of the birefringence data to infinite field strength, which gives
-0.023 for the optical factor. Earlier values for the optical factor of calf
thymus DNA and sonicated fragments obtained from electric birefringence

TABLE V
Optical Parameters of Various DNA Fragments in TE2 Buffer
Decay as
Base TI^^^ -6,Ica -6JCb -(r1 -72)
Sample Pairs a t High E (%) (deg/gg/ml) (deg/gg/ml) -(gl - g2) ( X 1021cm3)
B2 80 - - - - -
B3 98 100 0.27 0.35 0.026 1.5
B4 129 100 0.24 0.38 0.028 2.0
B5 161 100 0.24 0.34 0.025 2.5
B6 217 100 0.35 0.41 0.031 3.3
B7 257 87 0.32 0.38 0.028 3.4
B8 294 79 0.32 0.40 0.030 3.6
B9 389 61 0.32 0.40 0.030 3.6
B10 465 54 0.33 0.40 0.030 3.8
G2,Y2 628 35 0.26 0.33 0.025 2.9
G3 782 30 0.29 0.38 0.028 3.6
B12 830 24 0.30 0.37 0.028 3.0
Y4 940 22 0.30 0.36 0.027 3.2
Y5 1080 25 0.30 0.39 0.029 4.1
Y6 1314 33 0.32 0.39 0.029 6.6
PBR322 4364 10 0.31 0.36 0.027 6.4
a At high E.
From extrapolation vs 1/E.
422 STELLWAGEN

0.21 I
0 5
1IE i10’. kv-’Cm

Fig. 12. Typical example of the extrapolation of the specific birefringence to infinite field
strength for sample B8, 294 bp, in TE2 buffer: (O), 9.5 pg/ml; ( A ) , 11.2 pg/ml; (@), 10.0
pg/ml.

andlor electric field light-scattering measurements have ranged between

-0.016 and -0.0348,29,35736,75; optical factors calculated from flow bire-
fringence data are an order of magnitude l o ~ e r . l ~The , ~ reason
~ for this
discrepancy is not known, although it has been attributed2 to the necessity
of using the rotational diffusion constant, which is sensitive to flexibility
and polydispersity, in the flow birefringence calculation.
The optical factor of DNA can also be calculated from the birefringence
of oriented fibers. Using the value of An = -0.11 reported by Seeds and
W i l k i n ~for
~ ~DNA fibers in the B-configuration, the optical anisotropy
factor is calculated to be -0.023, in good agreement with the experimentally
determined values.
The optical factor can also be calculated from the Peterlin-Stuart
equation, Eq. (ll),using the birefringence of oriented fibers77and the re-
fractive index increment in aqueous solution, dnldc = 0.175 cm3at 633 nm78
to estimate the principal refractive indices of the DNA particles. The
Peterlin-Stuart value of the optical factor is calculated to be -0.0035, an
order of magnitude smaller than the measured value. This result occurs
because the second term in Eq. (ll),corresponding to the form anisotropy,
is nearly as large as the term corresponding to the intrinsic birefringence.
However, there is some evidence that the theoretical contribution to form
anisotropy is o v e r e ~ t i m a t e d .The
~ ~ value of the intrinsic anisotropy cal-
culated from the Peterlin-Stuart equation, neglecting form anisotropy
completely, is -0.020.
Tsvetkov46,80and Frisman et a1.81have estimated the optical anisotropy
of a DNA base pair by the method of tensor addition of bond polarizabili-
ties. The values obtained were -190 X and -150 X cm3, re-
spectively. Using the average of these values for (71- yz),the intrinsic
anisotropylbase pair, the optical anisotropy factor can be calculated from
Eq. (12). However, this relationship cannot hold for the DNA fragments
studied here, because Eq. (12) predicts that the optical factor should de-
 BIREFRINGENCE OF DNA FRAGMENTS 423

crease with increasing molecular weight (i.e., volume/particle), whereas

the experimental values are independent of molecular weight. If the vol-
ume/particle in Eq. (12) is assumed to be the volume occupied by a single
base pair, the optical factor is calculated to be -0.031, which is of the same
order of magnitude as the experimentally observed values. Harringtons2
has estimated the optical anisotropy/DNA base pair to be -129.3 x
cm3from flow birefringence data and the persistence length of DNA in 0.2M
NaC1. The corresponding value calculated for the optical factor, assuming
the volume/particle to be the volume occupied by a single base pair, is
-0.024.
In saturating fields (210 kV/cm), where nearly the whole population of
DNA molecules is oriented, the relative amplitude of the slow component
in the decay of the birefringence was roughly inversely proportional to
molecular weight, as shown in Fig. 4. Since the induced polarizability is
proportional to L 2 or M 2 (see below), it is the extended or stretched-out
molecules in each population of fragments which will interact first with the
electric field. Hence the concentration of the orienting species is not the
nominal DNA concentration but that fraction of the population which exists
as “fully extended” molecules. To calculate the optical factors corre-
sponding only to “fully extended molecules,” the observed optical factors
were multiplied by the relative amplitude of the decay of the slow compo-
nent of the birefringence in saturating fields (Table 11). Because the rel-
ative amplitudes were inversely proportional to molecular weight, the op-
tical factors corresponding to the orientation of “fully extended” molecules
also were inversely proportional to molecular weight, as predicted by Eq.
(12). From these optical factors and Eq. (12), optical anisotrapies/base
pair were calculated and are given in column 7 of Table V. The average
value for all fragments was (-3.5 f 1.5) X cm3. Without the values
for the two largest fragments, for which the amplitudes of 710ng were known
least precisely, the average value of (71 - 7 2 ) was (-3.0 f 0.9) X cm3.
This value is of the same order of magnitude as the values of -6.58 X
cm3 calculated by Rau and Bloomfieldl for the optical anisotropy/segment
of T7 DNA in 0.5 mM NaCl and the -4.3 X cm3 estimated by Har-
tingtons2 from a combination of flow birefringence data and the persistence
length of DNA in 0.2M NaC1. The values of (71 - 7 2 ) calculated from the
theoretical optical anisotropies/DNA base pair of T ~ v e t k o vand ~ ~Fris-
,~~
man et a1.81are -6.3 X and -5.0 X cm3, respectively.

Specific Birefringence at Low Field Strengths: Kerr Law

Behavior
At sufficiently low values of the electric field strength, the specific bi-
refringence of the various DNA fragments was proportional to E2, as can
be seen from the limiting slope of 2 in Figs. 3 and 4. The low-field portions
of the saturation curves are shown on an expanded scale in Fig. 13; Kerr
law behavior was observed for all fragments. The value of the electric field
424 STELLWAGEN

E? wlcm E2. rv/cm E2, hv/sm

Fig. 13. Dependence of the specific birefringence of the various DNA fragments on the
square of electric field strength. DNA concentration, ca. 10 pg/ml in TE2 buffer.

strength a t which the Kerr law was no longer obeyed was approximately
inversely proportional to molecular weight, as shown in Fig. 11.
In order to determine the limiting values of the specific birefringence
a t low field strength, values of 6/cE2were computed for each fragment and
extrapolated vs E 2 to E 2 = 0. These limiting values of 6/cE2are given in
Table IV; each represents an average of 2-7 separate determinations for
each fragment. The corresponding values of the specific Kerr constant
K,, calculated from Eq. (2) are given in the next column and are plotted
in Fig. 14 as a function of the number of base pairs in the fragment. The

10 I I

- K,,
lo5

I I
lo2 lo3 11
Base Pairs
Fig. 14. Dependence of the specific Kerr constant on the number of base pairs in the
TE2 buffer; ($I) TE8 buffer; (A)TE2 buffer
fragment. DNA concentration, ca. 10 pg/ml in (0)
+ 1 m M MgC12.
 BIREFRINGENCE OF DNA FRAGMENTS 425

specific Kerr constant was proportional to the square of the number of base
pairs (slope = 2) between 80 and 250 bp, leveled off between 250 and 1000
bp and then increased again. The same trends were observed in TE1
buffer, TElO buffer, and TE2 buffer plus 1mM NaC1, although in the latter
two cases the specific Kerr constants were 25-50% lower than observed in
TE2 buffer, as shown in Table 111.
The specific Kerr constants of the DNA fragments in TE2 buffer plus
1mM MgC12 were much lower than observed in TE2 buffer and increased
monotonically throughout the molecular-weight range covered (slope =
0.7). The reason for this different type of behavior is not known; as dis-
cussed above, the whole Mg2+-DNA interaction needs to be better char-
acterized before drawing conclusions about the effect of Mg2+on the po-
larizability of DNA. The optical factor has been shown to be independent
of Mg2+ ion concentration and equal to that observed in the presence of
monovalent cation^.^

Electric Polarizability of D N A
From a knowledge of the specific Kerr constant of DNA and the optical
factor calculated from the steady-state birefringence at infinite field
+
strength, the electric anisotropy factor, P Q, can be calculated from Eq.
(7) for each of the DNA fractions. Because the rise of the birefringence
of the smaller DNA fragments was accurately symmetrical with the decay
a t low field strengths (Fig. 101, the DNA molecules must orient by an in-
duced dipole mechanism. Therefore, at least a t low E , the permanent
dipole term in the electric orientation factor must equal zero and the whole
electric anisotropy term can be attributed to the induced polarizability.
The induced polarizabilities calculated for each fragment from Eq. (7) are
tabulated in column 6 of Table IV. Since the optical factor was indepen-
dent of molecular weight, the induced polarizabilities (all - a I ) follow the
same variation with molecular weight as K,, (Fig. 14).
For the smaller DNA fragments, the induced polarizability was pro-
portional to the square of molecular weight (and/or length), as shown in
the insert of Fig. 15. The leveling off and curvature at higher molecular
weights (see Fig. 14) can probably be attributed to the flexibility of frag-
ments larger than 250 bp.
To calculate the induced polarizability corresponding to the orientation
of “fully extended molecule^,'^ polarizabilities were calculated using the
optical factor corresponding only to the orientation of “fully extended”
molecules, i.e., multiplying the observed optical factor by that fraction of
the birefringence in saturating fields corresponding to the relative ampli-
tude of the slow component in the birefringence decay. The corresponding
polarizabilities, designated (q- cxL)*, are plotted in Fig. 15. Within
experimental error, the induced polarizability is proportional to the square
of the length of the orienting particle over the whole range of molecular
weights studied here. This length, calculated from the observed decay of
426 STELLWAGEN

1 1 1

0.1L
lo2 lo3 lo4
Length. A.
Fig. 15. Logarithmic plot of the dependence of the induced polarizability attributed to
the orientat,ion of whole DNA molecules (all - el)* on the length of the orienting particle.
DNA concentration, ca. 10 pg/ml in TE2 buffer. The drawn line has a slope of 2. Insert:
Dependence of the induced polarizability of the smaller DNA fragments on the square of the
number of base paidfragment.

the birefringence using the Broersma equation, may represent the total
distance over which the bound counterions move under the influence of
the electric field.

Comparison with Experimental Data

Hogan et al.,3 studying the electric dichroism of DNA restriction frag-
ments, also found the electric anisotropy factor to be proportional to L 2,
although they attributed the polarization mechanism to an apparent dipole
moment caused by anisotropic ion flow about the polyion. Stoylovs3 re-
ported that the electric polarizabilities of rigid rodlike fragments of tobacco
mosaic virus were also proportional to L 2.
Most of the values of the electric polarizability of DNA which exist in
the literature must be considered order-of-magnitude estimates because
most of the samples were sonicated or intact samples of calf thymus DNA,
with very poor control of the molecular weight. Weill et a1.8 calculated the
electric polarizability of sonicated calf thymus DNA to be 14 X cm3
from a combination of electric and flow birefringence measurements;
however, this value is uncertain because of the lack of correspondence of
 BIREFRINGENCE OF DNA FRAGMENTS 427

the optical factors determined by the two methods, as discussed above.

More recently, Sokerov and Weil12 used a combination of electric bire-
fringence, electric dichroism and polarized fluorescence in an electric field
to calculate the electric polarizability of a sonicated sample of calf thymus
DNA having an average length of 1200 A, intercalated with the dye acridine
orange. The value obtained for the polarizability was 3.5 X cm3,
about one-quarter that observed for B9,389 bp (1130 A). However, they
used DNA solutions about 10 times as concentrated as those used here, with
no determination of the possible concentration dependence of their results.
In addition, the length of their sonicated sample was estimated from the
initial slope of the field free decay of the birefringence at low field, with no
estimate of the polydispersity of the sample. The interaction with acridine
orange may also have affected the observed electric polarizability. Em-
onds-Alt et al.4 estimated the mean electric polarizability of calf thymus
DNA to be 22 X 10-32 F m2 (20 X 10-16 cm3) from the shape of the electric
birefringence saturation curves, assuming a pure induced dipole orientation
mechanism with three different polarizabilities. The problems with this
analysis have already been discussed.2 Ise et al.84estimated the polariz-
ability of calf thymus DNA to be 9.1 X cm3 from the low-frequency
dispersion of electric conductivity. However, there may be some uncer-
tainty in assigning this dispersion to the electric polarizability; Tricot et
al.85 have identified the high-frequency dielectric increment with the
electric polarizability determined from electro-optic data. However, it
should be noted that objections have been raised to this analysis2 and that
another explanation has been proposed for the high-frequency dielectric
dispersion of p~lyelectrolytes.~~ Finally, from the dielectric dispersion
of a low-molecular-weight(-450 bp) sonicated sample of calf thymus DNA
and theoretical equations for the dielectric increment, Vreugdenhil et al.s6
estimated the electric polarizabilities to be 20 X F m2 (18 X cm3)
and 130 X F m2 (120 X cm3) from the high- and low-frequency
dispersions, respectively. The polarizability calculated from the high-
frequency dispersion is about 50%higher than the value determined here
for B10,465 bp. However, the comparison should not be made too exactly,
since the sonicated samples of Vreugdenhil et al. contained fragments with
a fairly wide range of molecular weights, while sample B10 was a doublet
with an average molecular weight of 465 f 15 daltons.

Comparison with Theories of Electric Polarizability

Mandel’s equations7 for the anisotropy of polarizability of a charged
rodlike macromolecule is
n z 2e2L
(all - a d =
12kT
where n is the number of condensed but mobile counterions of valence z,
e is the electronic charge, and L is the length of the polyion. Equation (25)
428 STELLWAGEN

E2. k v / c m
Fig. 16. Comparison of the theoretical saturation curves of OYO with the experimentally
determined saturation curves of several DNA fragments. (a) B4,129 bp, 4.8 pg/ml in TE2
buffer, c1I2 = 0.17 cm/kV; (b) B6.217 bp, 8.9 pg/ml in TE2 buffer, p2 = 47, c1/2= 0.14 cm/kV,
b = 0.27 cm/kV; (c) High-field portion of the saturation curves of B9, B12, and Y6; the low-field
portions of the saturation curves are shown on an expanded scale in (dj and (e). (dj (0)B9,
389 bp, 16.5 pg/ml in TE2 buffer, b = 0.06 cm/kV; (ej Y6, 1314 bp, 7.6 pg/ml in TE2 buffer,
b = 1.9 cm/kV.

correctly predicts the L and 1/T dependence of the polarizability found

experimentally, but the further dependence of the induced polarizability
on M (through n )does not agree with the experimental results. In addition,
Eq. (25) predicts that the polarizability should be higher in solutions con-
taining divalent cations, whereas the opposite is observed (Table 111,and
Ref. 1). Other treatments of electric polarizability by Hornick and Weill,lo
Takashima,s8 and Ise et al.84also predict that the polarizability should be
proportional to L". The treatment by McTague and Gibbssg predicts an
approximate dependence of the polarizability on L a t low degrees of poly-
merization, and L 3 at high degrees of polymerization. The counterion-
fluctuation model for DNA,17 developed to explain the low-frequency di-
 BIREFRINGENCE OF DNA FRAGMENTS 429

I I I - I - ~-
20 30 40
P
\ O lo E2 ku/cm

I I I I I
0 1 2 3 4
E2, k v h m
Fig. 16. (continued f r o m the previous page)

I
0.7 - -

0.3 - -

0.1- -
0

0.03 I I
430 STELLWAGEN

0 5 10 15
E , kvtcm
Fig. 18. Typical plots of the orientation function versus E: (9) B5,161 bp, 14.7 pg/ml in
TE2 buffer; (A)B7,257 bp, 10.4 pg/ml in TE2 buffer; (0) Y6, 1314 bp, 11.1pglml in TE2
buffer.

Comparison with O'Konski-Yoshioka-Orttung (OYO) Saturation

Theory
According to the OYO saturation theory?? the shape of the birefringence
saturation curve as a function of E determines the ratio of the permanent
to the induced dipole moment of the orienting species, while the displace-
ment of the experimental curve on the horizontal axis of the theoretical
curve determines the sum of the permanent and induced polarizabilities.
Theoretical curves calculated for pure induced dipole orientation ap-
proximately describe the saturation behavior of the smallest DNA frag-
ments (5160 bp), as shown in Fig. 16(a) for B4,129 bp. The value of the
polarizability used to calculate the theoretical curve was 2.1 X cm3,
in excellent agreement with the value of 2.0 X cm3 calculated from
K,, and ( g l - g2). Polarizabilities obtained from the theoretical saturation
curves of the other small fragments also agreed well with the experimentally
determined values.
The qualitative shape of the birefringence saturation curves began to
change as the molecular weight increased above 161 bp (see Figs. 3 and 4).
Kerr law behavior was observed for a smaller and smaller fraction of the
total saturation curve, as shown in Fig. 17, causing the Kerr law region to
be shifted to lower and lower E (Fig. 11). Since complete saturation of the
birefringence occurred at approximately the same value of E for all samples,
the birefringence saturation curves became more and more spread out in
E. When the experimental saturation curves of these larger DNA frag-
ments were compared with the theoretical curves of OYO, they appeared
to exhibit increasing amounts of permanent dipole character, as shown in
Figs. 16(b), 16(d), and 16(e),with the saturation behavior of fragments B9,
 BIREFRINGENCE OF DNA FRAGMENTS 431

389 bp, and B12,830 bp, being best described by the theoretical curve for
permanent dipole orientation. The experimental saturation curve of
sample Y6,1314 bp, was even more spread out in E than the theoretical
curve for permanent dipole orientation, and the experimental points fell
above the theoretical curve a t high E, as shown in Fig. 16(c).
It seems unlikely that a homologous series of DNA fragments would or-
ient in an electric field by totally different orientation mechanisms de-
pending on the molecular weight of the fragment. In addition, the satu-
ration curve of sample B6,217 bp, is best fit by a theoretical curve for mixed
induced and permanent dipole orientation, as shown in Fig. 16(b), although
the rise of the birefringence is accurately symmetrical with the decay [Fig.
1O(b)], indicating pure induced dipole orientation. Therefore, the OY 0
saturation equations cannot be used to describe the saturation behavior
of DNA fragments larger than about 160 bp, probably because of the flex-
ibility of the larger fragments.
If the larger DNA fragments were progressively straightened by in-
creasing electric fields, the fraction of relatively stretched molecules in the
population would be increased. Since the polarizability is proportional
to the square of the length of the orienting species, one might expect
straightened molecules to interact increasingly more strongly with the
electric field with increasing field strength, causing saturation to be reached
more quickly than predicted by the OYO equations. However, the opposite
is observed; the saturation curves of the larger fragments are broadened,
i.e., more spread out in E than predicted by the OYO equations, suggesting
that increasingly shorter segments of the DNA molecules may interact with
the electric field as the field strength is increased. The appearance of
shorter components in the decay of the birefringence from high fields [Fig.
7(a)] and the decrease in apparent relaxation time with increasing pulse
length (Table I) are consistent with coiling of the larger DNA molecules
in high electric fields.

Comparison with Sokerou- Weill Saturation Theory

Sokerov and Weil12 have pointed out that Mandel’s formulation of the
anisotropy of polarizability is formally equivalent to a permanent di-
pole,
y = (n/12)1/2zeL (26)
where the terms have been defined above. Progressive saturation of the
induced polarizability at high fields26-2sleads to a limiting value of y at high
E,
y- = nzeLI2 (27)
Using Shirai’s comment2that the orientation function of a saturated in-
duced dipole should be integrated only between the limits of 0 and ir/2
(because the electrostatic energy for the orientations 8 and T - 8 are
432 STELLWAGEN

equal), Sokerov and Weil12developed new expressions for the orientation

function. If saturation of the induced polarization is reached at values of
E sufficiently low that pEIRT I3,
= p m E / k T = nzeLEf16kT (28)
and CP becomes linearly dependent on E.
T o see whether this type of analysis could describe the saturation data
of the DNA fragments between the Kerr law region and the region of total
saturation, the saturation curves were replotted vs E. A few typical ex-
amples are given in Fig. 18. DNA fragments I160 bp, which obeyed the
Kerr law over more than 60% of the saturation curve (Fig. 17), exhibited
no first-order dependence on E. Therefore, saturation of the orientation
occurred before saturation of the induced moment and the OY0 saturation
equations were obeyed [Fig. 16(a)]. However, the saturation curves of the
larger samples, after an initial region of curvature corresponding to the
low-field E dependence of the birefringence, all exhibited a significant
linear dependence on E which could be extrapolated to CP = 0 at E = 0.
From the slopes of these linear portions of the saturation curves, values of
the saturated induced dipole were calculated for the various fragments from
Eq. (29) and are tabulated in the last column of Table IV.
The saturated induced dipole was found to be constant for all fragments,
with an average value of 6400 f 300 D. This value is about three times
larger than the values obtained by Sokerov and Weill for sonicated calf
thymus DNA fragments containing intercalated acridine orange,2 but is
close to the values of 6000-6800 D calculated by them from other dichroism
data in the literature on DNA a l ~ n e . ~ , ' ~
From the definition of the saturated induced dipole, Eq. (as),one would
have expected it to be proportional to nL, i.e., approximately proportional
to L2. However, Eq. (28) was derived from Mandel's equation for induced
polarizability, which itself does not describe the dependence of the induced
dipole on length and/or molecular weight. Further theoretical studies of
the induced polarizability are needed.

I would like to express my appreciation to Prof. B. H. Zimm for the opportunity to work
in his laboratory; to Prof. R. Doolittle for the loan of the birefringence apparatus; to Dr. P.
J. Hagerman for assembling the birefringence apparatus, and writing the program for the
minicomputer and for helpful discussions; and to Prof. J. Donelson and Dr. J. Hartley for help
in the preparation of the restriction enzyme fragments. Financial support from NIH Grant
No. GM11916 and American Chemical Society Grant NP-150 is also gratefully acknowl-
edged.

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Received February 15,1980

Accepted July 25,1980