BIOPOLYMEItS VOL. 8, Pl’. 1-20 (l!)(i!

J)

Sedimentation, Diffusion, and Osmotic Pressure

of Sodium DNA in Salt-Free Solution

H E N R Y E. AUER* and Z. ALEXANDROWICZ, Polymer Departmerit,

W e i z m a n n Institute of Science, Rehovoth, Israel

Synopsis
The osmotic, sedimentation, arid diffusion coefficients of sonicated sodium DNA i n
aqueous solutions which were rendered rigorously free of salts have been obtained iii
order to study the polyelectrolyte effect on these parameters. Both native and heat-
denatured DNA’s were studied. All three parameters were forind to be independent
of concentration between 2 and 10 mmole/l. The osmotic coefficient gives the fraction
of counterions not immobilized by the polyelectrolyte potential, under conditions of
equilibrium. The value (ca. 0.18) obtained for this fraction agrees very well with that,
predicted by a theory of rodlike polyelectrolytes. A simplified model that permits the
computation of fractions of mobile ions from sedimentation and diff iision coefficients,
when applied to the experimental data, showed t,hat tinder these conditions of flow be-
tween 0.5 to 0.9 of counterions are not, immobilized. The experimental dat,a also showed
that for native DNA the translation frictional coefficient seems to have different values
in sedimentation and diffusion, which agrees with a finding reported for another salt-
free polyelectrolyte. For denatured DNA, however, the valiies were found to be equal;
no satisfactory explanation coiild be found for this difference of behavior.

INTRODUCTION
The solution properties of polyelectrolytes, in contrast to the usual
electrolytes, are very much affected by the pronounced asymmetry of the
component ionic species. The polyion is of an extended (often variable)
shape and bears numerous charges, fixed to the macromolecular chain,
while electroneutrality is assured by the presence of the stoichiometrically
equivalent number of oppositely charged counterions of low valency.
Ideal behavior would be observed if the polyion and each counterion were
independent kinetic units, free of the influence of the other particles
present. Due to the intense electrostatic interactions between them
however, such is not the case.
The first to study the thermodynamic parameters of polyelectrolyte
solutions was Kern.’ He concluded that the osmotic coefficient 4 of salt-
free solutions of poly(acry1ic acid) neutrnlized to various extents with
sodium hydroxidc decrc:Lsed strongly :LY the linear charge density on the
* Research Fellow of the Helen Hay Whitney Foundation. Present Address:
Department of Biochemistry, The University of Itochester School of hledicirie and
Deiitistry, Rochester, New York 14620.
1
2 AUER A N D ALEXANDROWICZ

chain increased and was considerably less than one, the value correspond-
+
ing to ideal behavior. I n addition, Kern found that was almost inde-
pendent of molecular weight and concentration. While the osmotic
coefficient for a polyelectrolyte may be determined from osmotic-pressure
measurements and from light-scattering studies, the counterion activity
coefficient (which may be considered thermodynamically identical to the
osmotic coefficient2) may be derived from measurements of half-cell
potentials and membrane potentials. It is generally found that values of
4 obtained from these various techniques agree one with another. Further-
more, patterns of behavior similar to that found by Kern for polyacrylate
solutions are followed by all polyelectrolytes. The osmotic coefficient
thus constitutes a valuable parameter, characteristic of the linear charge
density of a given polyelectrolyte and determining its behavior under a
variety of equilibrium experimental conditions.
A number of theoretical calculations, based upon solutions of the
Poisson-Boltzmann equation governing the polyelectrolyte potential, have
sought to describe the equilibrium properties of polyelectrolytes. Lifson
and Katchalsky3 derived an exact expression for for the case of a rodlike
polyelectrolyte, since for this model an analytical solution to the differ-
ential equation could be found. Subsequently, the predictions of the
theory were applied to many polyelectrolytes possessing varying degrees
of flexibility.2 It was invariably found, however, that the values of inter-
charge distance corresponding to the fully-extended state of the chain had
to be severely reduced in order to bring the calculated values of 4 into
accord with experiment. At the time, osmotic coefficients of a truly
rodlike polyelectrolyte were not available for unequivocal comparison
with theory.
When a polyelectrolyte is introduced into a system of electroneutral
translational flow, the polyion and counterion components migrate to-
gether. The observed frictional coefficient f may be expected to be com-
pounded of additive contributions from the polyiori and the z (monovalent)
counterions, so that to first approximation

f,- and f c representing the frictional coefficients of a single polyion and

counterion, respectively. It may be anticipated, however, that a con-
siderable portion of the counterions will be immobilized by the polyion, in
analogy to the equilibrium behavior of polyelectrolytes. The compound
frictional coefficient is thereby diminished, and may be expressed

n-here e represents the fraction of counterions not immobilized by the

polyelectrolyte potential. This simplified approach has been adopted by
Wall arid c o - ~ o r l i e r sand
~ ~ proved
~ to be very successful for interpreting
many flow systems.
 SODIUM DNA 3

Is the counterion immobilization in a system undergoing flow the same

as a t equilibrium, i.e., is 6 equal to C$? The question was recently examined
by Alexandrowicz and Daniel,6 who plotted values of 0, interpreted from
electrical transference or from sedimentation, versus 4, for a large number
of polyelectrolytes culled from the literature. (Unfortunately, the values
of C$ and e were rarely obtained in the same laboratories and on the same
samples.) The points so obtained are illustrated by circles in Figure 1.
They describe a broad arc lying decidedly above the line of slope unity,
along which 0 = 4. There seems little doubt, then, that in sedimentation
and electrical transference the fraction of free counterions is considerably
greater than under conditions of equilibrium. It seems established
beyond doubt that the mechanisms responsible for counterion immobiliza-
tion in equilibrium become modified in translational flow.
If so, do different systems of flow affect the immobilization to different
extents? I n such a case the values of 0 and hence off will depend on the
type of flow, and the time-honored assumption that frictional coefficients
in various translational flow systems are identical (on which basis, for
example, rests the Svedberg' equation connecting diffusion and sedi-
mentation), will not hold true for salt-free polyelectrolytes. Indeed, it
has been found that in salt-free solution the frictional coefficient of poly-
lysine hydrochloride in sedimentation turns out to be 1.8 times larger than
that in diffusion,8 which appears to be much larger than the experimental
error. Such disparity, if real, of course raises grave problems in the
systematic study of flow properties. I n this connection let us also mention
the diffusion potential across a boundary separating two salt -free solutions

Fig. 1. Plot, of 0 versus 4 for varioiis 1,olyelectrolyt.es:,).( (0) diverse polyelectro-

lgtes from sedimeirtat,ioii aiid elect r i d t ransfereiice, respect ively;6 ( 0 ) polylysiiie
hydrochloride from sedimeiitatioii;* (V),(V ) iiative high moleciilar weight I)NA from
sedirnentatio~i~' and electrical tra1isfereiice,4"respectively. Present results for 8 versiis
4 in salt-free soiiicated DNA solutions are: (A), ( A ) native DNA from sedimeutat,iori
atid diffusion, respectively; ( 0 )denatiired DNA from both sedimentation aiid diffusion.
4 AUER AND ALEXANDltOWICZ

of a polyelectrolyte a t different concentrations, which by the Nernst

equation should depend on the transference number t, of the polyion. It
has been sho\tq2 however, that when values of t, for sodium polyacrylate4
and sodium polypho~phate~ derived from electrical transference experi-
ments are introduced into the expression for the diffusion potential, the
predicted values are not in agreement with those actually measured,l0 once
again apparently beyond the limits of experimental error. This dis-
crepancy suggests that the frictional coefficients in electrical transference
and in diffusional flow are also different. Quite generally, therefore, it
appears that the frictional coefficients of polyelectrolytes are subject to
variation, depending on the flow process, but of course, such a far-reaching
finding needs to be further examined.
In order to further study some of the issues raised here, we have care-
fully examined thermodynamic and translational flow properties of the
system, sodium DNA-water, using DNA which had been sonically de-
graded to a molecular weight of about 500,000. Measurements of the
osmotic pressure, the sedimentation coefficient, and the diffusion co-
efficient were carried out with solutions which had been rendered rigorously
free of low molecular weight salt. Both native and heat-denatured DNA
szmples were examined. Our first objective was to check the Lifson-
Katchalsky theory for the osmotic coefficient of polyelectrolyte rods with
a substance which actually fits the model. Our second objective was to
compare the values of the frictional coefficients in sedimentation and
diffusion in order to determine whether they were perceptibly different.
In addition to the purely phenomenological determination, we tried also to
draw conclusions as to changes of ion immobilization and other possible
factors affecting the frictional coefficient in different flow systems.

EXPERIMENTAL
DNA
Calf thymus DNA from Worthirigton Biochemicals, Inc. was dissolved
i u standard saline citrate (SSC, 0.15M KaCl arid 0.015M trisodium
citratc) and further deproteinized by a few extractions with ch1oroform:iso-
arnyl alcohol (24 :1) solutiori." The DXA was precipitated with isopro-
paiiol, washed, arid immcdiatcly redissolved i n 0.05M sodium phosphate
buffer, pH 6.5, containing 0.002M EDTA. The DNA concentration was
2 mg/ml.
Sonication
Sonic irradiation of 3540 ml of the buffered DNA were carried out
under an atmosphere of in :L Raytheori 10 KHz sonic oscillator.
The sample cup was cooled by circulating ice water. Sorlication was for
pcriods of 10 inin, a t 0.9 to 1.1 A. The pH's and spectrophotometric
illeltirig curves a t 259 mp before and after soriicatiori were essentially
identical.
 SODIUhl DNA 5

Molecular Weight
JIolecular weights of the sonicated DNA samples were determined b y
measuring the intrinsic viscosity in SSC a t 25.00 f 0.04"C. The Cannon-
Ubbelohde viscometer used had a solvent efflux time of 24s sec. The
double-logarithmic curve of intrinsic viscosity versus molecular weight of
Eigner and Doty13 provided values for the molecular weight of the soni-
cated samples. These were corrected according to the recalibration of this
curve for low molecular weights implicit in the work of Cohen and Eisen-
berg.14 The molecular weight average finally obtained is, in general, more
complex than the weight average, which light s ~ a t t e r i n g ' ~yields,
, ~ * sincc
intrinsic viscosity is involved in the determination.
I n Table I the corresponding average numbers z of monomer residues per
molecule are set out for the various DKA samples. Samples IT-1 and D-1
represent cases where the products of two runs in the sonic oscillator,
having almost identical molecular weights, were combined and treated as a
single homogeneous sample, since the variance of the distribution of
molecular sizes produced by sonicationl* is much greater than the dif-
ference in molecular weights of the sonication runs so combined. Samples
D-1 and D-2, are, in fact, the same material: after performing one set of
diffusion and sedimentation runs on sample D-1, the entire batch was
recovered, concentrated, and redialyzed and was designated D-2.

TABLE I
Physical Characteristics of Sonicat,ed DNA Samples

Intrinsic z x 10-18
f,O

Designa- viscosity, (Aestimated g/sec-mole

tion State dl/g error) (.testimated error)

N- 1 Native 3.79 1400 f 5% 0.208 f 10%

N-2 Native 4.21 1510 f 570 0.219 f 10%
N-3 Native 5.87 1940 f 57, 0.261 f 10%
D-1
Denatured] 3 , 61 670 3= 570 0.030 107~
11-2 Denatured

* Measured on the native form, prior to denaturation.

Denaturation
Native sonicated DNA whose molecular weight had been determined was
dialyzed a few times against deionized water to remove most of the salt
present. The DNA, in the dialysis sac, was heated to 95°C for 1 hr,
cooled to room temperature over about 3 hr, and dialysis continued as for
samples of native DNA.
Salt-Free Solutions
Salt-free solutions of extreme purity were required. If the DNA solu-
tion is lO-*M in monomer residues, and if at equilibrium the value of 4 is,
say, 0.2, then the concentration of osmotically active ions is 2 X 10-3M.
 I n order to introduce less than 1% error by the presence of extraneous 1-1
electrolyte, the acceptable limit of contamination is 1 x lO-"dI salt.
The solutions of sonicated DNA in 0.05Jl sodium phosphate were con-
centrated to one-half or one-third the original volume. Ordinary cellulose
dialysis tubing was extracted \vith byarm sodium bicarbonate solution,
then with warm ethanol and finally Lvith warm distilled water. The con-
centrated DNA solution was encased in a dialysis sac, an air bubble also
being incorporated to ensure thorough mixing. The sac was introduced
into a capped Lucite cylinder holding about 300 cc and set to tumble a t
about rps a t 4°C. The dialysate was refreshed with cold distilled
deionized water, whose conductivity was less than 1 X reciprocal
ohm-cm-l. To check the efficiency of the dialysis procedure, sodium
chloride was introduced into the sac and its loss followed both conducti-
metrically and by means of chloride analysis on the dialysates. The limit
of detection of chloride was 0.04 pmole. The decrease in titratable chloride
paralleled the decrease in specific conductivity. The conductivities of the
last dialysates, which were equilibrated for 24-43 hr, were essentially
identical t o that of deionized water, and no detectable chloride was found.
Thus the salt concentration in the dialysate was always lower than 5 X
10-6M and should have been even less in the DKA solution itself due to the
Donnan effect. Imbibition of water considerably reduced the concentra-
tion of DNA during the course of the dialysis.
Proper caution was exercised to prevent contamination of the DNA by
salt subsequent to dialysis. All vessels, which were preferably of plastic,
were thoroughly rinsed with deionized water. All transfers arid manipula-
tions were carried out in the cold.
The concentration of the solutions of salt-free DNA was determined
spectrophotometrically, by using volumetric dilutions of the DNA into
cold SSC. An extinction coefficient, based on moles phosphate, of 6.53 X
lo3was employed.'j The DKA obtained upon completion of dialysis was
shown t o be fully native by carrying out melting curves on such dilute
solutions in SSC.
Dilute solutions of salt-free DNA were prepared by delivering cold
deionized water to the salt-free DNA produced by dialysis. A motor-
driven micrometer syringe fitted with a fine polyethylene capillary added
water at the rate of 2 pl/sec, while the DNA, maintained a t O"C, was being
rapidly stirred. Dilutions to about 1.5 to 2 X 10-3n'l were routinely made
and intermediate concentrations could then be prepared by appropriate
mixtures of the concentrate and dilution. Aliquots of the resulting salt-
free dilutions were transferred into SSC, on which melting curves were
carried out t o make sure that no denaturation had occurred.
Thermal Stability
Thermal stability of salt-free DNA was examined by conducting spectro-
photometric melting experiments between 0 and 40°C on salt-free DNA
solutions, in the absence of added salt. A set of quartz spacers provided
 SODIURI 1 )NA 7

Temperature, “ C
Fig. 2. Thermal denaturation of sample N-1 in salt-free solution meawred by irirrease
of relative absorbance: ( 0 )1.0 X 1 0 P mole P/L; ( 0 )11.5 X mole P/l.

path lengths of 1.0, 0.2, or 0.05 mm, as required, for the highly concen-
trated solutions. Two representative melting curves are presented in
Figure 2. As expected, the solution of higher concentration undergoes
thermal denaturation a t a higher temperature than does the solution of low
concentration. This is presumably a manifestation of a self-protectivc
effect due to the increased “ionic strength” provided by the higher con-
centration of free counterions a t the higher DXA concentration, and is in
direct analogy to the increased protection to thermal denaturation afforded
by increasing concentrations of added salt. 16*
Since a t 5°C a DYA solution with a concentration as low as 1 X 10-36f
remains completely native (Fig. 2), all our experimental determinations
were conducted a t this temperature on solutions of concentration greater
than this limiting value.
Diffusion
Diffusion measurements were made on an instrument which had been
constructed in our laboratory by ;\Iohan18 according to the outlines of
L o n g s ~ o r t h ,employing
~~ Rayleigh interference optics with the 5461 A
mercury line. The temperature was maintained a t 5.3 =t 0.01”C. To
prevent denaturation of native DNA by diffusion into distilled water, the
solvent side n-as filled with dilute D S A solution. Solutions of denatured
DKA, however, were allowed to diffuse into distilled water.
Diffusion coefficients D were calculated by two methods designed to
examine the dependence of D on concentration c. Both were programmed
for electronic computation. First, the method of BoltzmannZ0permits the
determination of D as a function of c from single experiments:

D(c) = (- 1/2t)(rZx/tlc),
/r: xdc‘

Here, x is distance (in centimeters) in the cell, while c is evaluated from the
number of fringes and the refractive increment, dn/cZc.
* Our results disprove the unexpected finding” that higher concentrations of DNA
are more susceptible to denatiuatioii ripon removal of salt than are low cwnceritratiotis.
8 AUER AND ALEXANDROWICZ

Second, independence of D from concentration can be tested by the

method of L ~ n g s w o r t h ,i ~
n ~which the coordinates of several pairs of
points in each fringe diagram, on opposite sides of the median point, are
required to fit values predicted from the error function. If all pairs of
points meet this criterion,1Y2" I ) is indeperldcllt of c and may be easily
calculated.
I n all cases, a zero time correction was applied to account for the finite
dimension of the boundary a t the beginning of the experiment. The true
value of D , DO,can be obtained as the intercept in a plot of D versus l / t . 2 1
Our results for D , since they were obtained by applying the methods of
Boltzmann and Longsworth, are identified as being values of Dzm, in the
terminology of Gosting. Therefore the D values found are weight-average
values. 22
The diffusion apparatus was checked by determining the diffusion
coefficient of sucrose. The results agreed very well with the literature
value.23
Sedimentation Velocity
Sedimentation velocity experiments were carried out with a Spinco
model E ultracentrifuge and a Beckman model E ultracentrifuge. Angular
velocities were 59,780 or 60,000 rpm on the respective instruments. The
rotor was maintained a t 5 f 0.3"C. The cells were 12 mm Kel-F double-
sector cells which were converted to the capillary-flow synthetic boundary
type. Due to rapid diffusion of the boundary, photographs were taken a t
intervals of 2 miri or less. The schlieren peaks obtained in differential
sedimentation runs (see Results) were always perfectly symmetrical. In a
differential sedimentation run on a preliminary preparation of DSA, in
which eleven photographs were evaluated, the sedimentation coefficients
obtained using root-mean-square peak positions was indistinguishable from
that found using simply the maximum peak positions. Consequently, in
the work reported here, only the latter were measured. From these data
the sedimentation coefficients were evaluated by the moving boundary
method as the least-squares slope of plots of In z versus t. The values of s
so obtained correctly represent polydisperse samples; they are in fact
weight-average sedimentation coefficient^.^^

Osmotic Pressure
Osmotic pressure was measured by the concentration osmometer tech-
n i q ~ e . ~ I5n this procedure, a n unknown polymer solution is made to
undergo osmosis against a series of concentrations of a standard polymer of
known osmotic pressure. Interpolation to the point where zero volume
flow between chambers occurs yields that concentration of the standard
with the same osmotic pressure as the unknown solution.
The polymer used as ultimate osmotic standard was a poly(ethy1ene
glycol) sample, Carbowax 6000, of the Union Carbide Chemical Co.
 SODIUM DNA 9

The osmotic pressure of this material as a function of concentration in the

range of 0-3 g/dl was determined by light scattering on solutions in
0.2M KaC1. (As has been shown elsewhere,z6the scattering intensity of
this substance is the same in salt free as in salt-containing solutions.)
Solutions were clarified by extensive centrifugation a t 20,000 rpm and the
scattering measured on ;t S@ca light-scattering photometer, with the use
of polarized light a t 5461 A. No angular dependence of scattering was
found. A curve of osmolality mP+,where m p is the molality of the solution,
versus concentration is shown in Figure 3. Three points extracted from
previous resultsz6are shown for comparison.
I n the osmometric runs, this polymer as well as a second sample of
poly(ethy1ene glycol), Carbowax 2031, were variously used as standards.
The osmolality of the latter was measured in the concentration osmometer,
using cellophane membranes, against Carbowax 6000. I n the range of
interest, the osmolality of Carbowax 2031 was 0.685 that of an equal con-
centration by weight of Carbowax 6000. Both polymers were exhaustively
dialyzed against deionized water and lyophilized prior to use in experi-
ments with DNA. The standard solution was prepared by weight, the
dry polymer being absolutely not hygroscopic.
Some difficulty was encountered in obtaining membranes which would
rigorously exclude a leakage of the standard polymer into the D N A cham-
ber. Nost of the membranes used were of cellulose acetate cast in our
laboratories according to the method of L ~ e b .These
~ ~ membranes were
successfully employed in osmotic experiments involving as standard
Carbowax 2011, or the denatured D K A which was used to measure a
native D N A sample. A membrane cut from thick-gauge dialysis tubing,
which had been boiled in sodium bicarbonate and subsequently in ethanol,
was used in experiments involving Carbowax 6000 as standard.

C,gm / 100 ml

Fig. 3. Osmolality, mP4, in moles/l. of Carbowax 6000 in 0.20iM NaCl, as a function

of weight concentration of polymer; ( 0 )selected values taken from ref. 26.
10 AUEl: AND ALEXANDROU'TCZ

The experiments were carried out in the cold room, a t 3-Ti"C. (The
concentration osmometer method is little affected by temperature changes
if the temperature remains the same on both sides of the membrane.) The
osmometers were gently agitated during the runs, which took about 1 day
with the Loeb membranes, and about 4-6 days with the cellophane mem-
branes.
Material reclaimed from diffusion experiments (made saline, recon-
centrated, and redialysed to the salt-free state) was subsequently em-
ployed in the osmometric runs. Thereafter, however, it had to be dis-
carded due to the possibility of contamination by the Carbowax standards.

RESULTS
Osmotic Coefficients
The results of the osmometric experiments are presented in Table 11.
The third column gives the concentration of Carbowax 6000 which was
isoosmotic with the DNA sample. (In the osmometry of sample N-3, the
standard polymer employed was sample D-2.) The osmolality of the
DNA solution is then known by reading off the curve of lcigure 3, and the
osmotic coefficient 4, given in the fourth column, is the ratio of this con-
centration t o the stoichiometric monomolar concentration of DNA present.
I n addition to the standard deviations given in Table 11, an estimated error

TABLE I1
Osmotic Coefficients of Native and Heat-Denatured DNA at 5°C

Concn of
osmotic
DNA Conc. standard,
Sample M x 103 g/100 ml 6 (A std. dev.)

N-1 4.18 0.511 0.169 (&4% est,imated)

N-2 1.94 0.226 0.155
4.55 0.564 0.171
7.90 0.936 0.174
Mean 0.167 f 370
N-3 3.00 1.96* 0.154
5.53 3.6gn 0.157
6.91 4.89* 0.165
8.90 5.898 0.156
Alean 0.158 f 2%
D-2 3.52 0.595 0.238
4.67 0.716 0.220
7.24 1.18 0.254
9.33 1.35 0.232
Mean 0.236 f 37'

8 The osmot,ic standard is DNA sample D-2. Concn in M X lo3.

 SODITJRf DNA 11

of 37, arising from the calibration of the standard polymer must be added.
It should be noticed that, in the ranges examined, 4 is in effect independent
of concentr:~tioti (up to about 9 X 10-3Al), and, for native DNA, of
apparent molecular \\eight. This is in agreement n-ith the general be-
havior of polyelectrolytes discussed earlier.

Diffusion Coefficients
Both of the computational methods employed to examine the relation-
ship between the diffusion coefficient and concentration showed that D, is
independent of c over the entire range in question. The numerical results
of the two methods were in excellent agreement. They are set out in
Table 111. The concentrations of the two solutions on either side of the
boundary are indicated in the second column. The values of D, for
native DNA are seen to be independent of apparent molecular weight.
The value of D, for sample K-2 was determined from a synthetic boundary
run in the ultracentrifuge under conditions of negligible centrifugal flow.
It is seen to differ slightly from the other values obtained for native DNA.

TABLE I11
Iliffrisiori Coefficients of Native and Heat-Denatured DNA at 5.3"C

D, X lo6, cm2/sec
Sample Concri range, M X 103 ( f std. dev.)

N-1 1.2-15.2 3.46 f 3%

N-2 1.8- 7.9 3.30 f 5%
N-3 1.5-13.5 3.52 f 1%
I)-1 0- 9 . 0 2.89 f 2%
D-2 0- 9 . 4 2.94 f 270
~-

Sedimentation Coefficients
The sedimentation boundaries were formed by means of a synthetic
boundary cell. I n some cases, for native DNA, the boundary was formed
between two solutions of different concentrations. The sedimentation
coefficient then determined was actually a differential sedimentation
coefficient,24 which is strictly valid a t the arithmetic mean of the two
concentrations. The remainder of the runs with native DNA were made
with boundaries formed against water. As a result, a small amount of
denatured DNA formed at the centripetal edge of the boundary, due to
diffusion into water. This was visible as a small peak. The denatured
DNA, however, sediments more slowly than does the native DNA and so
does not penetrate the boundary. Furthermore, the motion of the peak
in sedimentation describes the flow in the entire plateau region.28 Conse-
quently the denaturation has no effect on the value of s,. All the runs
with denatured DNA were carried out by forming a synthetic boundary
against water. The values of the sedimentation coefficients obtained,
12 AUER ANII ALEXANIIROWICZ

TABLE I V
Sedimentation Coefficients of Native and IIeat-Denat wed I)NA at 5.3%

s,, X loL3,sec
Sartiple COIICII, ill x 103 (fstd. dev.)

N-1 1 . 2 - 9.04 1.181

1.2 -15.2 1.072
5.51-11.3 0.961
11.3 -15.2 1.122
15.2 1.14X
&lean 1 .O9G + 457
N-2 I .8 1.016
1.8-7.1) 1.129
7.9 1.155
>lean 1.100 + 4y0
N-3 2.16 1.073
8.38 1 ,088
10.4 1.036
11.5 1.027
15.7 1.068
Mean 1.048 f l y (
D-1 3.60 0.736
5.40 0.942
7.20 0.902
9.00 0,925
Mean 0.876 4~ 570
11-2 2.82 0.976
5.26 0.856
7.52 0.858
9.40 0.940
Mean 0.908 f 370

corrected to 5.3"C, are given in Table IV. For the differential sedimenta-
tion runs, the concentrations of both solutions are indicated in column 2.
The value of s, for every sample is found to be independent of concentra-
tion of polyelectrolyte, in the range examined. I n addition, there is no
dependence of the s, values of native DNA on the apparent molecular
weight of the sample. The result that in the absence of salt both L), arid
s, show negligible dependence on concentration agrees with prediction.

DISCUSSION
Osmotic Coefficients
The osmotic coefficients of native and denatured DKA which appear in
Table I1 are depicted in Figure 4. Also shown for the sake of comparison
are the activity coefficients of the sodium counterion in solutions of sodium
DNA, as determined from measured membrane potentials by Ascoli
 SODIUM DNA 13

I I I I I I I I I
06 -

-8-
02

- - - - oO . O
t 1 2 3 4 5 6 7 8 9
)

c, M I I 18'

Fig. 4. Osniotic coefficients niid activity coellicieiits of sodirrm I>NA: ( 0 )YN,, of

iiative L)NA;29 (m),
(0) ~ 21.5"C of native arid denatured DNA, r e ~ p e c t i v e l y ; ~ ~
Y N a1
(X), (+) present resiilts for a t 5°C of native and denatured DNA, respectively.
C$

et al.29.30and by Lyons and K ~ t i n . ~The

l measurements of Lyons and
Kotin were carried out a t 21.5"C, while our data were obtained a t 4°C;
presumably Ascoli and co-workers also worked a t room temperature.
The three sets of measurements are in pronounced disagreement. I n
the present case, considerable care was taken to remove as much protein
contamination as possible from the commercial sample of DNA used in
the work. Preparation of the DKA from media containing citrate or
EDTA ensured the removal of divalent ion impurities. Furthermore, our
experimental technique scrupulously avoided the introduction of ex-
traneous ionic components. We note lastly that our results yield low
values of 4 and show essentially no dependence of 4 upon DNA concentra-
tion, in accordance with the behavior observed with diverse dilute poly-
electrolyte We consider, therefore, that the present de-
termination of the osmotic coefficients of the native and denatured forms
of sodium DNA are the most reliable to date.
The electrostatic free energy of rod-shaped polyelectrolyte molecules has
been computed by Alfrey et al.32and by Lifson and K a t ~ h a l s k y . ~The
osmotic coefficient 4 obtained upon differentiation of the electrostatic free
energy with respect to volume is given by3

4 = (1 + B2)/2X (3)
Here, p is an implicit function of X (for higher A, p2 < 0) and of the volume
fraction of polymer. X characterizes the linear charge density, and is
given by X = e2/DbkT, where e is the unit electronic charge, D is the
macroscopic dielectric constant of the solvent, b is the distance between
the fixed charges, and k is Boltzmann? constant.
12or native DKA, b is taken as 1.7 A, while the radius of the molecular
cylinder, needed to evaluate the volume fraction, is taken as 9.7 A.33 The
11 AUER AND ALEXANDROWICZ

resulting value of X is 4.25. The value of p2 when the DNA concentration

is 2 X 10-3M is -0.41, giving for C#J a value of 0.17. If the DNA concen-
tration is set equal to 10 X 10-3M, p2 equals -0.61, and C#J is computed to
be 0.19. It is seen that these values of C#J calculated for concentrations
which span the range employed in the present work are in very good agree-
ment with the experimental results, both in their magnitudes and in their
virtual independence from concentration. So far, the theory of Lifson
and Katchalsky has been shown to agree with data on various flexible-chain
polyelectrolytes only if b, hence X, is treated as an adjustable parameter
fitted to allow for deviations from the fully extended rod.2 Our study on
DNA tests the theory with a truly rodlike polymer, i.e., X is known. The
agreement of the calculated values of C#J with experiment provides sub-
stantiation for the validity of the Lifson-Katchalsky theory. (Spe-
cifically, the agreement between theory and experiment supports the use of
the macroscopic value of D in evaluating X.)
The discrepancy between our results and those of Ascoli et al. and of
Lyons and Kotin, noted above, cannot be explained by the fact that the
measurements were made a t different temperatures, since the product DT,
and hence X, is essentially independent of temperature.
When native DNA is denatured, the double-helical structure is dis-
rupted. If a single-stranded deoxyribonucleate chain were completely
extended, the intercharge distance b would be about 7 8. X is then 1.0, and
p2 is -0.18, when the concentration is 10 X 10-36f. When these values
are introduced into the Lifson-Katchalsky theory, a value for C#J of 0.57 is
predicted. This is clearly too high to accord with the experimental results,
and indicates that the actual charge density on the chain is considerably
greater. The adjusted value of b turns out to be about 2.4 8, making X
equal 2.9 and p2 about -0.49. From these results, it is seen that denatured
DNA behaves like other flexible chain polyelectrolytes, though the cor-
rection factor for X is relatively high,2 possibly indicating a quite compact
conformation of the chain in salt-free solution. This could be due to the
regeneration of a considerable fraction of hydrogen bonds between comple-
mentary bases in a nonspecific I n support of this contention,
it was found that melting curves obtained directly on salt free solutions of
the denatured DKA a t very short optical path lengths showed very broad
transitions, extending from 5 to 80°C. This was indicative of the re-
generation of 507, of the hydrogen bonds under the conditions of our
experiments.
Frictional Coefficients
I n order to conipare the frictional coefficient in a sedimentation experi-
ment Jb with that in a diffusion experiment j(i,let us define the empirical
parameter
RSD = SJSd (4)
 SODIUM DNA 15

If we substitute for the frictional coefficients the generalized forces required

to maintain unit velocity in translational flow,s eq. (4) becomes

\ ,

-
-
M,(l - iip)D,
RT&,
where A f p , ill,,,,and 8 are, respectively, the molecular weight, the residue
weight, and the partial specific volume of the neutral polyelectrolyte, p is
the density of the polyelectrolyte solution, s, and D, are the apparent
sedimentation coefficient and the apparent diffusion coefficient, respec-
tively, of the polyelectrolyte. b?r/dm, is the derivative of the osmotic
pressure with respect to the molar concentration. For a salt-free x-valent
polyelectrolyte, of osmotic coefficient 4, b?r/brn,is equal to RT&. x is of
course also the number of residues per macromolecule, so that M , = M,,/x.
For the sodium salt of DNA, M,,, = 331. I n salt-free solution the values
for B are, for native DNA, 0.499 cc/g f 1%,15 and for denatured DNA,
0.494 cc/g f 3%.
Since the values of the three experimental parameters +, D,, and s, for
each sample of DNA are independent of concentration, the values of RsD
determined from them are likewise not dependent on solute concentration.
This of course applies to that range of concentration in which all three
parameters have been obtained, which in practice is delimited by the con-
centration range for +. Since D, and s,, were determined over more ex-
tensive concentration ranges than 4 and were found to be independent of
concentration, we may suppose that 4, and therefore RSD, will also be
independent of concentration for the entire concentration range studied.
I n Table V, the value of R s D as calculated for each sample by eq. ( 5 ) is
presented, together with the concentration range defining its applicability.
It is seen that for every sample of native DNA, the ratio of frictional
coefficients is greater than one, beyond the limits set by experimental
error. We wish especially to emphasize the results for sample N-3. The
range of rigorous applicability is broad. The derived value of RSDis very

TABLE V
for~ Native and Heat-Denat rired DNA Calciilated from
Values of f i s ~
Osmotic Pressure, Free Diffnsion, arid Sedimentation Velocity

Sample Coricli raiige, M X lo3 Z i s ~(f estimated error)

N-1 4.18 1.34 f 0.19 (=k14%)

N-2 1 .!14-7.00 1.20 f 0.20 ( f l S % )
N-3 2.8:<-8.!I0 1.52 f 0.12 (+ 8%)
D-1 3.52-9.33 1.02 f 0.17 ( f l S % )
1)-2 3.52-9.33 1.00 f 0.15 ( f 1 4 % )
16 AUER AND ALEXANDROWICZ

large, 1.52, while the estimated error is low, 8%. We conclude, then, that
the frictional coefficient of native DNA in salt-free solution in sedimenta-
tion is greater than it is in free diffusion, over a fairly extensive range of
polyelectrolyte concentration.
In contrast to the behavior of native DKA, the values of R s D for the two
samples of denatured DNA are unequivocally equal to unity. (Since
samples D-1 and D-2 are actually the same preparation, the values of 4 for
D-2 were used to compute R s D for D-1.) We conclude from the results
that the frictional coefficient of denatured DKA in salt-free solution in
sedimentation is experimentally indistinguishable from that in free diffusion.
Could these findings be explained by a variation, with the system of flow,
of the counterion immobilization? According to the simplified model, the
friction of a polyelectrolyte molecule in the absence of salt is taken to be
additively compounded of the polyion and mobile ion frictions, so that
[see eqs. (1’)and (4)]
RSD= (fp- + eszfNa+)/(S,- + edzfNa+) (6)
Here fp- is the frictional coefficient of the DNA polyion and fNa+ of the
sodium ions, while 0, and & are the fractions of the mobile counterions in
sedimentation and diffusion, respectively. Daniel and Alexandrowicz,s
while discussing their work on salt-free polylysine hydrochloride (PLHCl),
have hypothesized that R ~ may D differ from unity for the following
reason. The application of an external field, as occurs in sedimentation
but not in diffusion, possibly modifies the degree of counterion immobiliza-
tion. If so, 8, is expected to be larger than e d , while Od and the osmotic
fraction 4 might be taken as equal one to the other, since both are repre-
sentative of the random motion of the ions, with no external force applied.
Thus, from the data for es and 4 in PLHCl one calculates RSD5 2 (only
an upper bound can be given since f p + of salt-free PLHCl is only known to
be larger than or equal to the value found in the presence of salt). This
prediction fully agrees with the value of R s D = 1.8 found experimentally.8
Now the main contribution to the friction of both native and denatured
DKA is due to the counterions, since 0zfN.c >>fp- (see the following section).
Therefore, R S Dis in effect given by the ratio &/&[eq. (S)]. Hence, adopt-
ing the hypothesis just described, we take e d = 4 and estimate R s D from the
ratio es/4 (see next section for evaluation of the 0’s presented in Table VII).
This gives R s D equal to about 3-4, for both native and denatured forms of
DNA. Experimentally, however, we find the much lower value R S D=
1.3-1.5 and the absolutely unaffected value R S D= 1, for the native and
denatured forms, respectively!
One could argue, of course, that the modification of the frictional
coefficient of :L salt-frec polyelectrolyte molecule depends on its shape.
We suppose here that native DKA is rodlike, denaturcd DXA is coillike,
and I’LHC1 presumably consists of very stiff, short coils.8 However, it is
difficult to envisage a mechanism for modifying the frictional coefficient
by which the most rodlike molecule, native DNA, would constitute the
 SODIUM DNA 17

intermediate case, giving an R ~ valueD larger than unity but much smaller
than computed from Os/tp.
Let us continue to speculate on the possible origins of this apparent
anomaly. We observe that any mechanism producing an alteration in
the frictional coefficient might well arise from an orientation of the poly-
electrolyte molecules in salt-free solution, under the influence of an external
field. If so, fs, and hence s,, should depend on the centrifugal field in-
tensity. We have therefore studied the effect on s, of varying angular
velocity, using a new sample of sonicated native DNA. Unfortunately
the experimental conditions of our original study could not be reproduced,
and only some preliminary low-accuracy experiments have been carried out.
The results are set out in Table VI. Tentatively fitting the results with a
straight line dependence of s, upon the relative centrifugal field (using
least squares and omitting a suspect value s, = 1.637), we find that as the
field decreases toward zero, s, tends to about 0.7 X sec. Recalcu-
lating RsD accordingly [eq. (31, we find R S D 2 at low field intensity.
The result, if credible, shows that in the absence of an orientation effect,
RsD of native DNA approaches more closely the value expected from
Os/& ( = es/tp). This is generally analogous to what has been found with
I’LHC1. Clearly, however, this still does not answer the question why R S D
is equal to one with denatured DKA. Summing up: the contradictory
evidence gathered so far for RSDin salt-free polyelectrolyte systems remains
baffling, unless of course it is attributable to experimental error. Let it be
added that determining s, and D, in salt-free solutions is very difficult and
admittedly of little practical importance, as opposed to determinations in
the presence of salt which permit the molecular weight to be evaluated.
Still, because of the great theoretical importance attaching to a possible
variation of frictional coefficients with flow, further study of the unanswered
questions is certainly needed.
Counterion Immobilization in Flow (e)
In the preceding section the presentation of the experimental frictional
coefficients was purely phenomenological (R ~ Dand
) the simplified model of

TABLE VI
Dependence of the Sedimentation Coefficient of Native DNA on
Relative Centrifugal Field

Angular velocity, rpm Relative centrifugal field sp X sec

40,000 1.053 0.870

40,000 1.053 0.853
48,000 1.516 1.104
52,000 1.779 1.052
56,000 2.063 1 .33!J
60,000 2.369 1,637
60,000 2.369 1.257
64,000 2.695 1.114
18 AUElt AND ALEXANDROWICZ

additive contributions to friction in salt-free solutions [eq. (l’)]was applied

only when trying to explain the significance of the results. Now let the
model be adopted straightaway and used in presenting the data. Thus
let us take618
fs 3 d ‘ m ( 1 - fip)/sp = esZfN.+ + fp- (7)
and
fd RTzd/Dp = 0dZfN.c -I- fp- (8)
Here 0, and Od, as mentioned previously, are the fractions of mobile ions in
sedimentation and diffusion,respectively. f~ a+ ( e 2 . 1 X 1Ol5 g / s e ~ - m o l e ~ ~ )
is obtained from conductivity experiments with inorganic sodium salts.
Because of constant shape of the native D N A ion f,- in salt-free solution
should be equal to the value f,”measured in the presence of salt. Thus
from diffusion data36 (extrapolated t o zero polymer concentration) , for
a native D N A sample of molecular weight 820,000 we find f,” equal to
0.305 X 1OI8 g/sec-mole. The values of r i for samples N-1, X-2, and
N-3 given in Table I were derived from this value and the dependence37of
fpo on z 2 I 3 . (Using the Riseman-Kirk\~ood~~ equation for long rods leads
to values 10% lower, which for the present purpose is immaterial.) The
results show that the friction of the native D N A ion contributes very little
to the total friction in the above equations. The value of the frictional
coeficient of the denatured D N A ion in salt-free solution is unknown.
However, we have measured frictional coefficients in the presence of salt,
finding fpo of sample D-2 to be 0.03 X 10l8(see Table I), from a diffusion
experiment carried out on a hydroxymethylated material (to prevent
aggregation39) in 0.2M SaC1. Now the lowering of the effective ionic
strength, from 0.2M NaCl to 20.014M concentration of the mobile ions12
gives rise to considerable chain expansion. Yet the increase of the trans-
lational frictional coefficient of the sample considered certainly cannot
exceed one order of magnitude, whereas even such an increase leaves f,-
negligibly small in the equations above. We conclude therefore that Os and
ed may be evaluated without bothering too much about the exact value
off,-.
Values of 0, and ed calculated with eqs. (7) and (8) for native arid de-
natured D N A are set out in Table VII, values of n being reproduced by thc

TABLE VII
Fractions of Free Counterions for Native and Heat-Denatured DNA in
Sedimentation (&), Diffusion (ed), and by Osmotic Pressure (6)

Sample e, (festimated error) ed (.testimated error) 9

N-1 0.655 f 754 0.476 f 13% 0.169
N-2 0.652 f 790 0.494 f 15‘/0 0.167
N-3 0.693 + 4(h 0.434 f 9% 0.158
11-1 0.900 f 9% 0.887 f 9% 0.236
1)-2 0.870 f 7% 0.872 f 9yo 0.236
 SODIUM DNA 19

way of contrast. The results are also added to the plot of 0 versus 4 for
various polyelectrolytes (Fig. 1) and are seen to essentially follow the
general behavior. (Not entirely, for with PLHCl 0, was founds to be
distinctly larger than &, both having been determined from slopes in the
presence of salt, while e d turned out t o be equal to 4 ; i.e., es > & 'V 4 was
found. Here, however, we find 0, > > 4 and 0, N > 4 with the
native and denatured DNA, respectively. This difference in behavior
reflects however what we have already discussed in connection with R ~ D . )
For comparison we have also evaluated 0 of a high molecular weight
native Na DNA. Applying the method of Wall and co-workers4 to their
electrical transference data, Inman et aL40 found 0 N 0.40; applying the
method of slopes in the presence of salt8 to the sedimentation data of Stern
and Atlas4' (using points a t 1.0, 0.2, 0.1, and 0.0lM NaCl but discarding
the point a t 0.00lM) we find 0 N 0.50. All these values, which agree
roughly in their magnitudes but lack detailed consistency, prove the
approximate validity of the simple model used in treating sedimentation
and diffusion data in this section. Let the finding be stressed that the
frictional contribution due to the mobile counterions, e Z f N n k , exceeds by a t
least ten times the contribution due to the polyion, f P - , arid accordingly,
that S , and 1/D, in salt-free solution are an order of magnitude smaller
than in the presence of salt. Hence the description accorded by this
simple model, even if approximate, affords an explanation for very large
effects.
We wish to thank Professor Aharotr Katchalsky for his active aiid coritiriried interest
this investigation. I h . G. R. Mohaii's iriterisive instruct,iori in the art, of diffitsiometry
iti
coiisiderably facilitated much of the work. Mr. Ariel Lustig's generous assistance in
some of t,he sedimentat,ion atialyses is greatly appreciated.
We wish to thank Mr. Yeheskiel Haik for performing measiiremeiits of fi of denatured
IINA in identical fashion w in ref. 15.

References
1. W. Kern, Z . Phys. Chem., A181, 249 (1938); A184, 197, 302 (1939).
2. A. Katchalsky, Z. Alexandrowicz, and 0. Kedem, i i i Chemical Physics of I o n i c
Solutions, B. E. Conway and R. G. Baradas, Eds., John Wiley, New York, 1966, pp.
295-346.
3. S. Lifsori and A. Katchalsky, J . Polyni. Sci., 13, 43 (1954).
4. J. 1i. Huizenga, P. F. Grieger, aiid F. T. Wall, J . Ainer. Chevi. Soc., 72, 2636
(1!)50).
5. J. It. Huixenga, P. F. Grieger, aiid F. T. Wall, J . A n m . Chem. Soc., 72, 4228
(1950).
6. Z. Alexaridrowicz arid E. Ilaiiiel, Biopolpiers, 1, 447 (1963); Hiopolymers, 6,
1500 (1968).
7 . T. Svedberg arid K. 0. Pedersoti, 71/14f,'~/racvntr<'i/qv,Oxford Utiiversity Press,
Lottdoii, 1940.
8. EL 1)aniel atid Z. Alexaiidrowicz, Hiopol~yniers,1, 4 3 (I!)6:3).
!). F. T. Wall aitd It. TI. Ihremtis, J . A t ~ i e r C
. h e w . Soc., 76, 868 (1954).
10. C. Bolre, V. L. Cresceiizi, A. 11.Liyiiori, atid A. Alele, Trans. Par. Soc., 55, 1975
(1959).
11. A l . G. Sevag, D. B. Lackmarl, aiid J . Smoleiis, J . Biol. Chem., 124, 425 (1958).
20 AUEIl AKU ALI3SANUlIOWICZ

12. P. Doty, B. B. McGill, and S. A. Itice, Proc. Natl. h a d . Sci., 44, 432 (1958).
13. J. Eigner and P. Doty, J . Mol. Biol.,12, 549 (1965).
14. G. Cohen and H. Eisenberg, Biopolymers, 4,429 (1966).
15. G. Cohen and H. Eisenberg, Biopolymers, 6, 1077 (1968).
16. W. F. Dove and N. Davidson, J . Mol. Biol.,5,467 (1962).
17. H. J. Lin and E. Chargaff, Biochim. Biophys. Acta, 145, 398 (1967).
18. G. R. Mohan, Ph.D. Thesis, Weizmann Institute of Science, 1967.
19. L. G. Longsworth, J . Amer. Chem. SOC.,74, 4155 (1952).
20. J. Crank, The Mathematics of Digusion, Oxford University Press, London, 1956.
21. L. G. Longsworth, J . Amer. Chem. Sac., 69, 2510 (1947).
22. L. J. Costing, Adv. Prot. Chem., XI, 429 (1956).
23. L. J. Godrig and M. S. Morris, J . Amer. Chem. Sac., 71, 1998 (1949).
24. H. K. Schachman, Ultracentrifugation in Biochemistry, Academic Press, New
York, 1959.
25. Z. Alexandrowicz, J . Polyrri. Sci.,40, 113 (1959).
26. Z. Alexaridrowicx, J. Polyiri. Sci., 40, 107 (1959).
27. S. Loeb and G. It. Nagarj, Chem. Abstr., 66,5687p (1967).
28. S. Goldstein, Proc. Roy. Soc., A219, 151 (1953).
29. F. Ascoli, C. Botre, V. Crescerizi, A. Mele, and A. hl. Liquori, Nature, 184, 1482
(1959).
30. F. Ascoli, G. Botre, and A. M. Liquori, J . 14401. Biol., 3, 202 (1961).
31. J. W. Lyons and L. Kotin, J . Amer. Chem. SOC.,86,3634 (1964).
32. T. Alfrey, P. W. Berg, and H. Morawetz, J . Polym. Sci.,7,543 (1951).
33. R. Langridge, D. A. Marvin, W. E. Seeds, H. It. Wilson, C. W. Hooper, M. M. F.
Wilkins, and L. I>. Hamilton, J . Mol. Biol.,2, 38 (1960).
34. D. 0. Jordan, The Chemistry of Nucleic Acids, Butterworths, London, 1060.
35. H. S. Harned and B. B. Owen, The Physical Chemistry of Electrolyte Solutions,
Reinhold, New York, 1958, for example.
36. R. Cecil and A. G. Ogston, J . C h e m SOC.,1948, 1382.
37. H. Morawetx, Macromolecules in Solution, Interscience, New York, 1065, pp.
271-273, for example.
38. J. Riseman and J. G. Kirkwood, J . Chem. Phys., 18,512 (1950).
39. L. Grossman, S. S. Levine, and W. S. Allison, J . Mol. E'iol., 3, 47 (1961).
40. R. B. Inman and D. 0. Jordan, Biochim. Biophys. Acta, 42,421 (1960).
41. K. G. Stern and S. M. Atlas, J . Biol. Ckem., 203, 795 (1953).

Received October 24, 1968

Revised February 26, 1969