Hydrophobic Polyacids in Denaturant Solutions

n
0

-1

8 2

-I
"

2
(Log

KL Icalc

Figure 6. Experimental values, (log K L ) for~ alkali

~ ~ metal
~ ions
with triphenylphosphine oxide in tetrahydrofuran at 25' vs.
values, (log KL)calcd, calculated using eq 20 based on an ioridipole model corrected for surrounding polarizable solvent.
scale of our plot. The value of B found corresponded to a
value of the radius of the sphere b = 6.9 X 10-8 cm. The
plot of (log &Jexpt us. values of (log KL)calcd calculated
from eq 19 using the B value (= 0.695 X 1016 cm-2) found
above appears in Figure 6.
The radius of the sphere containing cation and ligand
(or solvent molecule) required to fit our data to eq 20 (or

142'7

19) is very reasonable. The diameter of a molecule of THF

may be estimated from models to be -5.5 A. This would
lead to an estimate of b of 6.5 to 7 A. For Ph3P0, the effective b value would be expected to be somewhat larger.
We conclude that ion-dipole interaction, modified to
include the effects of surrounding polarizable solvent, can
account for the values of cation-ligand association constants of the alkali metal cations with triphenylphosphine
oxide in tetrahydrofuran solvent. Of course, we cannot say
that we have proven that these are the only factors that
affect the process. As we have pointed out above, we have
neglected several factors in our equation which are known
to be involved: ion-induced dipole interaction, dipoledipole repulsion in the complex, and van der Waals dicpersion interaction. We have advanced what we consider
to be good reasons for neglecting the contributions due to
these factors. In order to assess the relative importance of
these terms we have neglected, it would be advantageous
to have cation-ligand association data for several other di pole ligands with the alkali metal ions. We hope to accumulate such information in the future.
We further conclude that the effect of surrounding POlarizable solvent on the energy of interaction between ions
and solvent molecules in the first solvation layer28 should
be taken into account in efforts to calculate energies of
solvation.21929

Conformational Transitions of Hydrophobic Polyacids in

Denaturant Solutions. The Effect of Urea
P. Dubin' and U. P. Strauss"
School of Chemistry, Rutgers University, The State University of New Jersey, New Brunswick, New Jersey 08903
(Received November 27, 1972)
Publication costs assisted by the United States Public Health Service

The effect of urea on the charge-induced transition from hypercoiled to extended conformation of alkyl
vinyl ether-maleic acid copolymers has been investigated in 0.04 M sodium chloride solutions in the
presence and absence of 5 M urea. Potentiometric titrations indicate that the free energy of stabilization
of the uncharged compact state relative to the hypothetical uncharged extended state is reduced upon
addition of urea from 310 to 120 cal/mol of residue for the polyacid with butyl side chains, and from 1070
to 830 cal/mol of residue for the hexyl copolymer. Intrinsic viscosity results obtained with the ethyl and
butyl copolymers a t various degrees of dissociation show that urea expands the molecular dimensions of
the copolymers in both hypercoiled and extended conformations. Urea appears to destabilize the hyper.
coiled state by enhancing the solvent affinities of the hydrophobic side chains as well as the polar back.
bone of the polyacids.

Introduction
Considerable interest has arisen concerning the made of
action of denaturants on biological marcromolecules, inasmuch as such processes are thought to elucidate the forces
stabilizing the native state. The denaturant activity of
such widely varied substances as alcohols, detergents, and

urea and guanidine compounds is currently rationalized in

terms Of the Several types Of interactions which maintain
secondary and tertiary structure in biopolymers. Studies

(1) Present address: Dynapol Corporation, Palo Alto, Calif. 94304.

The Journal of Physical Chemistry, Voi. 77, No. 11, 1972

1428

P. Dubin and U. P.

Strauss

with small molecule model compounds, chemically similar

to protein moieties, have been of great value in identifying
plausible sites of interaction and estimating corresponding
thermodynamic contributions to the denaturation process? It is nonetheless quite difficult to specify exactly
the mechanism of action of a denaturant, such as urea,
which is primarily effective a t very high concentrations.
For example, considerable evidence exists from model
compound studies to the effect that urea (1) interacts
strongly with peptide backbone groups, thus weakening
interpeptide hydrogen
and (2) enhances the solubility of nonpolar groups, thus reducing hydrophobic interactions, either by altering the bulk properties of the
solvent or through more localized effect~,~,5,6
It is difficult
to assess the relative significance of such effects in studies
with natural biopolymers.7,s
An alternative approach to this problem may be available through the use of synthetic macromolecular model
compounds. In our laboratory, for example, we have studied hydrolyzed copolymers of maleic anhydride and alkyl
vinyl ethers which undergo conformational transitions resembling those occurring in the denaturation of biopolymers. When their alkyl side chains contain four or more
methylene groups, these copolymers exist a t low pH in
highly compact conformations with intrinsic viscosities
similar to those of globular proteins.SJ0 These "hypercoiled" states, stabilized by cooperative hydrophobic interactions among side chains, undergo reversible conformational transitions to expanded polyelectrolyte configurations upon progressive neutralization of the carboxylic
acid g r o u p ~ . ~ J O
We present here an exploratory study of the effects of
concentrated urea on such copolymers and their conformational transitjons. Our aims are to investigate whether
there are, indeed, effects similar to those observed with
proteins, and if so, whether and to what extent any such
effects can be ascribed to action on specific chemical
groupings, such as the hydrophobic groups belonging to
the side chains and/or the more hydrophilic backbone.
Attempts at such a differentiation appear promising with
our synthetic copolymers, because the length of their hydrophobic side chains may be varied, a procedure not generally feasible with natural biopolymers. Specifically, we
shall consider the effect of 5 M urea on the difference in
free energy between the uncharged compact and (hypothetical) expanded states of the butyl and hexyl vinyl
ether copolymers in dilute sodium chloride solution. This
free energy difference is obtained from potentiometric titration data for these polyacids, in conjunction with those
of the nonhypercoiling homolog, the ethyl vinyl ether copolymer.lO These data are supplemented by results obtained under the same conditions giving the effect of 5 M
urea on the dependence of the intrinsic viscosity on the
degree of dissociation for the ethyl and butyl vinyl ether
copolymers.

Preparation of Polymer Solutions. All studies were performed in 0.04 M NaC1, either with or without 5 M urea.
In order to avoid degradation, polymer stock solutions
were prepared in the dark at 4" by stirring a weighed
amount of the dry anhydride form of the polymer with an
amount of NaOH solution equivalent to the total carboxylic acid content of the polymer, neutralizing with HC1,
and, finally, adding sufficient water and NaCl to make
the solution 0.02 N in carboxylic acid and 0.08 M in NaC1.
Prior to titration or viscometry, an aliquot of the refrigerated stock solution was mixed with an equal volume of
either water or freshly prepared 10 M urea. Polymer concentrations were determined from titration equivalence
points.
Potentiometric Titrations. Titrations of polymer solutions and appropriate solvent blanks were performed as
previously describedlO under nitrogen and at 30, using a
Radiometer pHM26 p H meter equipped with Radiometer
G202c glass and K4016 calomel electrodes. Titrant, 0.200
N NaOH, was added with a 2.0-ml Gilmont micrometer
buret. The value of the degree of dissociation CY (defined
as unity at the equivalence point of the first acid group),
corresponding to a desired pH, was determined from the
volumes of added titrant at that pH and a t the first equivalence point, corrected for the volume of 0.200 N HC1 required to bring a blank to the desired pH from the pH of
the equivalence point.1 It was assumed that the activity
coefficient of hydrogen ion is the same in polymer and
blank solutions.
Viscometry. Viscosities were measured in a CannonUbbelhode dilution viscometer a t 30" by successively adding appropriate volumes of solvent adjusted to the pH of
the initial polymer solution. The use of solvent as diluant,
as opposed to external dialysis solution, affects the slope
of the reduced viscosity-concentration plots, but not the
values of the intrinsic viscosity obtained by extrapolation
of those plots.11 Because of acid-catalyzed hydrolysis, solutions containing urea at low pH exhibit a slow pH drift
(ca. +0.1 p H unit/hr) resulting in a gradual increase in
the measured viscosity of the polymer solution. When the
pH drift was' small, either because the pH was not very
low and/or the measurements were made rapidly, the p H
of the final, most dilute solution was used to obtain the
value of CY corresponding to the extrapolated value of the
intrinsic viscosity. In order to obtain reliable measurements at the lowest pH values, and to verify the other
data, the following procedure was employed to maintain
constant pH throughout the measurement. A sample of
polymer solution identical with that introduced into the
viscometer was placed in the (thermostated) titration vessel and diluted with solvent simultaneously along with the
viscometer sample. Constant pH was maintained in the
duplicate sample by intermittent addition of 0.200 N HC1,
and identical volumes of HC1 were added to the dilution
bulb of the viscometer. With this procedure, the pH of the

Experimental Section
Materials. The preparation and characterization of the
copolymers of maleic anhydride and ethyl vinyl ether (AVI), butyl vinyl ether (B-11), and hexyl vinyl ether (C)
have been described previously.lO Mann Ultrapure urea
was recrystallized from hot ethanol. Fischer Certified
0.200 N sodium hydroxide and hydrochloric acid solutions
were used as titrants. All water was deionized and doubly
distilled.

C. Tanford, Advan. Protein Chem., 24, 1 (1970).

Y.Nozaki and C. Tanford, J. B i d . Chem., 238,4074 (1963).
D. R. Robinson and W. P. Jencks, J. Amer. Chem. SOC.,87, 2462
(1965).
D. B. Wetlaufer, S. K. Malik, L. Stoller, and R. L. Coffin, J. Amer.
Chem. SOC.,86,508 (1964).
W. Bruning and A. Holtzer, J. Amer. Chem. SOC.,83,4865(1961).
J. R. Warren and J. A. Gordon, J. B i d . Chem., 245,4097 (1970).
T. T. Herskovits, H. Jaillet, and 8.Gadegbeku, J. B i d . Chem., 245,
4544 (1970).
P. Dubin and U. P.Strauss, J. Phys. Chem., 71,2757 (1967).
P. Dubin and U. P.Strauss, J. Phys. Chem., 74,2842 (1970).
U. P. Strauss, J. Polymer. Sci., 33,291 (1958).

The Journal of Physical Chemistry, Vol. 77, No. 11. 1973

Hydrophobic Polyacids in Denaturant Solutions

I

1429

mm

-u -u =
0.45

40

n
I

0.80

5.0,
A

t0.14y

-*
Y

0.175

0.050

fl.10

0.165)

0.010

c,eq. I;'

4.0
I

0.5

1.0

a
Figure 1. Potentiometric titration data at 30", plotted according
to eq 2: (0)ethyl, ( A ) butyl, and (U)hexyl copolymers in 0.04
M NaCl (ordinate on right): ( 0 )ethyl, (A)butyl, and (H)hexyl
copolymers in 5 M urea 4- 0.04 M NaCl (ordinate on left).
final solution in the viscometer usually differed from the
initial value by less than 0.02 p H units. The total amount
of HC1 added was always too small to have a measurable
effect on polymer concentration or ionic strength. For urea
solutions above p H 5 , viscosity measurements could be
made in the usual way, as no p H drift was observed.

Results
Potentiometric Titrations. The p H of a poly(monoprotic
acid) is given by the well-known equation12J3

where bGion io the change in the excess free energy of the

polyacid accompanying an incremental change in the degree of dissociation, and KO is the intrinsic ionization constant, obtained by extrapolating the left-hand side of eq 1
to a = 0, at which point the second term of the right-hand
side of eq 1 vanishes. The analogous equation for a poly(diprotic acid) is1O
pH 4- log y = pKlo

+ (RT In lOF1(aGiOn/da) (2)

where
112

and where a is defined as unity a t the equivalence point

of the first acid group, and Kl0 and Kzo are the first and
second intrinsic ionization constants of a diprotic acid residue.
Titration data for the ethyl, butyl, and hexyl copolymers, in 0.04 M NaCl with and without 5 M urea, are presented in Figure 1 in the form of pK10 + (RT In 10)-1.
P G i o n / b a ) , obtained from eq 2, against a. By means of a
procedure described previously,1 pK10 and pKz0 are
found to be 3.8 and 6.6, respectively, for all three copolymers in the absence of urea. The corresponding values in
the presence of urea are found to be 4.4 and 7 . 2 . A similar
decrease in acid strength in the presence of urea has been

Figure 2 . The dependence of the reduced viscosity on polymer

concentration at varying degrees of dissociation: (0)ethyl copolymer in 0.04 M NaCI; ( A ) butyl copolymer in 0.04 M NaCI;
(Q, 0 , 0 ) ethyl copolymer in 0.04 M NaCl 4- 5 M urea (procedures 1, 2, and 3, respectively, see text); (A,V I A ) butyl copolymer in 0.04 M NaCl 4- 5 M urea (procedures 1, 2, and 3 ,
respectively).
noted for a number of simple dicarboxylic acids.14 The
procedure for extrapolating the curve for the butyl copolymer in the presence of urea is based on the assumption
that the values of pKIO and pK20, respectively, are identical for all three copolymers, and that the titration curves'
for the butyl and hexyl copolymers coincide at low values
of U. These assumptions are supported by previous titration data in pure water,1 as well as by our present findings in the absence of urea.
The regions of negative slope in the titration curves of
the butyl and hexyl copolymers in both solvents correspond to transitions from hypercoiled to expanded conformations.lO The standard state free energy change per
mole of residue for the (hypothetical) conformational
transition a t zero charge, AGtO, is proportional to the area
between the curve of the copolymer undergoing the transition and that of the ethyl copolymer in the same solvent.
The details of and justification for this method of determining AGtO have been given previously.lO The value of
AGtO for the butyl copolymer, expressed in cal per mole
residue, is 310 f 10 in the absence of urea, and 120 f 20
in the presence of urea. The corresponding values for the
hexyl copolymer are 1070 f 30 and 830 f 30. These error
limits encompass the range of reasonable extrapolations of
the data to a = 0. The areas between the curves are found
to be rather insensitive to the extrapolated value of pK10.
It is noteworthy that these AGtO values are considerably
more precise than those obtained by a similar procedure
for the helix-coil transition of ionizable polypeptides, in
which case the titration curve for the random coil conformation is not experimentally accessible.15
Viscosity. Several representative extrapolations to infinite dilution of viscosity data for the ethyl and butyl copolymers in the two solvents are shown in Figure 2 . In
order to facilitate comparisons between the two copoly(12)A. Katchalsky and J. Gillis, Red. Trav. Chim. Pays-Bas, 66, 879
(1949).

(13) A. Arnold and J. Th. G. Overbeek, R e d . Trav. Chim. Pays-Bas, 68,

192 (1950).
(14) M. Levy and J. P. Magoulas, J. Amer. Chem. SOC., 64, 1345
(1962).
(15) See, for example, T. V. Barskaya and 0 . B. Ptitsyn, Biopolymers,
IO, 2181 (1971).
The Journal of Physical Chemistry, Vol. 77, No. 17, 1973

1430

P. Dubin and U. P. Strauss

'

62

6.4

d6

0:8

Ib

1'2

Figure 3. Intrinsic viscosity as a function of a for ethyl and

butyl copolymers in 0.04 Iw NaCI, with and without 5 M urea.
Symbols identified in Figure 2. Inset shows A a vs. a for ethyl

(0)
and butyl (A)copolymers (see text).

mers of different monomer weights, concentration units

are equivalents liter-I, each monomer residue contributing 2 equiv of carboxylic acid.
The slopes of the reduced viscosity plots for both polymers in 0.04 M NaCl are negligibly small a t low charge,
and increase with a , presumably as a result of enhanced
intermolecular interactions. Since the diluting solvent was
not in dialysis equilibrium with the polymer solution, the
observed slopes may be lower than those corresponding to
the true Huggins' constants, if one component is preferentially absorbed by the polymer.ll Such interactions may
explain the negative slope of the butyl copolymer in urea
a t low a.
Semilogarithmic plots of intrinsic viscosity against a
are presented in Figure 3. This type of plot was chosen in
order to facilitate the empirical extrapolation to a = 0. It
is of interest that comparable data for polyacrylic and
polymethacrylic acids16 are also found to conform to linear semilogarithmic plots in the region of low a.

Discussion
Inspection of the appropriate areas of Figure 1 reveals
immediately that the free energies of stabilization of the
uncharged butyl and hexyl hypercoils are considerably reduced in the presence of 5 M urea. In the case of the butyl
copolymer, AGtO is reduced by 190 f 25 cal/mol (about
60%), while the corresponding decrease in AGtO of the
hexyl copolymer is 240 f 35 cal/mol (about 22%).
The effect of denaturants on the conformational stability of the native state of proteins has frequently been analyzed in terms of the behavior of small molecule model
compounds.2 In such treatments, it is customary to assume that hydrophobic peptide side chains are withheld
from the solvent in the native state and exposed upon denaturation. The presumably additive contribution of one
such group to the overall denaturant effect on the free
energy of the conformation change then corresponds to its
free energy of transfer from pure solvent to denaturant solution, which may be estimated from appropriate model
compound partition or solubility measurements.
Let us examine the consequences of applying an analogous procedure for the case of the butyl and hexyl copolymers of this study. The values of AGtO result from several
The Journal of Physical Chemistry, Vol. 77, No. 11, 1973

contributions, which include the hydrophobic interactions

among alkyl side groups, other interactions of the various
groups with one another and with the solvent components,
and conformational changes of the polymer chain. If we
assume additivity of the interactions involving the two
extra methylene groups of the hexyl copolymer, one-half
the difference in the AG,O values of the butyl and hexyl
copolymers, both obtained in the same solvent, should
represent lGCHzO,the free energy of transfer of a methylene group from the hypercoiled form to the extended
form, in the chosen solvent. We find AGCHz' to be 380 f
15 cal/mol in the absence of urea, and 355 f 15 cal/mol
in the presence of urea. If we assume further, as is frequently done in the analogous treatment of denaturant
effects on proteins, that in the compact conformation the
environment of the methylene groups of the hydrophobic
side chains is independent of the solvent, we obtain from
the difference in the l G C H 2 0values a value of -25 f 20
cal/mol for the transfer of methylene groups from 0.04 M
NaCl to the same solvent containing 5 M urea. For com. , the
~
solubility of
parison, the data of Wetlaufer, et C Z ~for
various hydrocarbons in water and in concentrated urea
may be interpolated to give a value of -60 f 10 cal/mol
for the same process, and those of Nozaki and Tanford,3
for solubilities of leucine and glycine in water and aqueous
urea, lead to a value of -50 f 10 cal/mol. Our result is of
the right magnitude. However, this may be somewhat fortuitous in view of the assumptions implicit in the course
of the calculations. For instance, the effect of urea on the
intrinsic viscosity of the butyl copolymer a t low values of
a, to be discussed below, throws some doubt on the assumption of nonaccessibility to solvent of the hydrophobic
side chains in the hypercoiled conformation. The implicit
assumption that the hydrophobic side chains are completely exposed to the solvent in the random coil conformation may also be questioned. These considerations
suggest that the agreement may in part be due to a cancellation of neglected effects, and that therefore in comparisons of this type applied to biological or other synthetic macromolecules caution should be exercised in
evaluating the significance of apparent agreement of this
sort.
Further information about the effects of urea on the
conformations of these copolymers may be obtained from
the intrinsic viscosity results shown in Figure 3. The intrinsic viscosity is a measure of the molecular dimensions,
which, in turn, depend on the interactions of the macromolecules with the solvent. Whereas the potentiometric
titration method as applied here yields only the effect of
urea on the difference in free energies of the hypercoiled
and hypothetical extended states at zero charge, the intrinsic viscosity should allow an estimate of the effect of
urea on the copolymers in their actual states a t both zero
and finite charge. The data show that urea raises the intrinsic viscosity of both ethyl and butyl copolymers over
the whole range of a investigated. Since the butyl copolymer is in its hypercoiled form a t low values of a and in its
random coil form a t high values of a , and the ethyl copolymer is in its extended form under all conditions, we see
that urea significantly enhances the solvation of both
compact and extended forms. However, a meaningful
quantitative interpretation of the effects of urea on the
two forms from a numerical comparison of the intrinsic
(16) I. Noda, T. Tsuge, and M. Nagasawa, J. Phys. Chem., 74, 710
(1970).

Hydrophobic Polyacids in Denaturant Solutions

viscosity changes appears impossible, primarily because of

the lack of adequate theoretical expressions relating the
viscosity of the hypercoiled conformation to the free energy of interaction with the solvent.
Nevertheless, a somewhat more limited quantitative interpretation of the results may be obtained by means of
an alternative procedure for which no explicit mathematical relationship involving the intrinsic viscosity is needed.
In this procedure the intrinsic viscosity is employed essentially as an indicator for comparing the effect of urea on
the macromolecular dimensions with that brought about
by a change in the degree of dissociation without urea. At
any value of a , the change from 0 to 5 M urea leads to a
certain increase in the intrinsic viscosity, as shown by the
vertical displacement between the no urea and urea
curves of Figure 3. At not too high values of a (see below)
an identical intrinsic viscosity increase may be brought
about by the addition of an appropriate amount of sodium
hydroxide (in the absence of urea). The resulting increase
in the degree of dissociation, denoted by A a , is given by
the horizontal distance between the appropriate urea
curve (at a ) and no urea curve (at a
101)of Figure 3,
and is plotted as a function of a for the two copolymers in
the inset of that figure.
The increase in [v] accompanying an increase in a is
due to an enhancement of the solvent affinity of the carboxylate groups attached to the polymer backbone. If
urea, like NaOH, affected the solvent affinity of the backbone region alone, 1 a would be independent of alkyl side
chain length. In fact, Aa is observed to be greater for the
butyl than for the ethyl copolymer, from a = 0 to cy = 0.4,
beyond which the method is not applicable because of the
~ ~ difference in Aa for
maxima in the [v] us. a C U I V ~ S .The
the two copolymers represents clear-cut evidence that

1431

urea enhances the solvent affinity of the hydrophobic side

chains. On the other hand, the relatively large value of
for the ethyl copolymer indicates that urea also enhances
the solvent affinity of groups close to or part of the polymer backbone. The slight decrease of l a with increasing
a may reflect a relative change in the solvent affinities of
carboxylic acid groups and carboxylate groups, in favor of
the former, brought about by the addition of urea. Such a
preference is indicated by the observation that the dissociation of carboxylic acids in aqueous solution is generally
diminished by the addition of ~ r e a . 1 ~
Concentrated urea thus reduces the stability of the hypercoiled relative to the random coil conformation by a
combination of effects which involve enhancements of the
solvent affinities of both kinds of conformations. Intramolecular interactions of the hydrophobic side chains as
well as of the groups located in the immediate domain of
the polymer backbone are affected. In view of the complex
interdependence of these interactions with one another
and with the macromolecular dimensions, a quantitative
resolution of the effects of urea into additive contributions
of the various chemical groups appears to be unwarranted
with the data thus far available.

Acknowledgment. The support of this research by

grants from the United States Public Health Service
(Grant KO.GM 12307) and from S. C. Johnson and Son,
Inc., is gratefully acknowledged. We also wish to thank
Sylvia Taylor for expert technical assistance.
(17) Such maxima, which are observed for these poly(diprotic acids)
with sodium but not with tetramethylammonium ion as the counter..
ion, may be attributed to the specific binding of sodium ion to the!
polyacids above (Y = l.i8,19
(18) A. W. Schultz and U. P. Strauss, J. Phys. Chem., 76,1767 (1972).
(19) A. J. Begala and U. P. Strauss, J. Phys. Chem., 76,354 (1972).

The Journal of Physical Chemistry, Voi. 77,NO. 11, 1973