Effect of the cations sodium, potassium

and calcium on the interaction of

hyaluronate chains: a light scattering
and viscometric study
J. K. Sheehan, C. Arundel and C. F. Phelps
Department of Biological Sciences, University of Lancaster, Bailrigg, Lancaster LA 1 4 YQ, UK
(Received 9 December 1982; revised 10 March 1983)

Light scattering and viscometric studies have been carried out on two preparations, A and B, of rooster comb
hyaluronate. Sedimentation rate studies have also been performed with A. Light scattering measurements in 0.2 M
KCI for preparation A gave a molecular weight of 3.3 × 106 and for B, 1.0 x 106. In (0.1-0.3) M NaCl similar
measurements gave a particle weight for A of (4.4-6.4)× 106 and for B (1.7-2.8)× 106. In 0.066 M CaCl,
molecular weight values of 9.5 x 106 for A and 1.7 × 106 for B were obtained. Thus in the presence of Na + and
Ca2 + ions aggregates of chains persisted into dilute solution. Measurements by light scattering on A and B in 4 M
guanidinium chloride gave values in the same range as those obtained in 0.2 M KC1. Sedimentation rate studies on
A gave values oflO.3 Svedbergs in 0.2 M KCI and 12.2 Svedbergs in 0.2 M NaCl and 0.066 M CaCl,. The shear
dependence of the viscosity was studied using a conicylindrical viscometer at shear rates between 0.5 and 20 s- I.
Preparation A in 0.2 M KCI and NaCl yielded values for (qw/c)c--~O of 5000 and 7100 ml g - i respectively in
keeping with the tendency to aggregate. The behaviour for preparation B was similar. In 0.066 M CaCl 2 there was
a marked dependence of viscosity on shear speed below I 0 s- l for all concentrations and the value of(qJc)c--*O at
0 s- l for preparation A was 7700 ml g - 1 while at a shear rate of 8 s- 1 (qsp/C)C_..~ 0 = 5000 ml g - l. Similar effects
were found for preparation B and the data suggest associations of chains disruptable by weak shear forces. The
increase in viscosity with concentration in the presence of 0.066 M CaCL was much less than in the presence of
KCI or NaCl, suggesting that the Ca 2+ had a marked effect on the 'rigidity' of the molecules in solution. A
viscometric titration experiment with C a 2+ showed that a level of O.02 M CaCl 2 in 0.2 M NaCl was sufficient to
produce the change in viscosity presented above and that significant perturbations of the viscosity were present at
0.005--0.01 M CaCI,.

Keywords: Viscosity; hyaluronate; light scattering; sodium; potassium: calcium

Introduction medium-sized oligosaccharide fragments ( ~ 6 0 disac-

Extensive physicochemical solution studies have been
made on hyaluronate in dilute solution 1 - 5. The resulting
model for hyaluronate in solution is that of a stiff random
coil. Low angle X-ray and viscosity data 6 have been used
to describe the shape in terms of a worm-like coil with a
persistence length around 4 nm. Most of these studies
have employed Na + as the counterion and no systematic
study of the effect of different counterions on dilute
solution properties has been attempted. The effects of
various counterions, in particular, Na +, K + and C a 2 +, on
the conformations and interactions of hyaluronate chains
in the solid state have been described 7-11. It has been
shown that these ions generate different conformations in
the solid state which react differently to such stimuli as
pH, humidity and temperature. In this study the size,
shape and interactions of hyaluronate molecules in
solution have been examined in the presence of Na +, K +
and Ca 2 +. The intermolecular associations and rheolo-
gical properties of sodium hyaluronate solutions have
recently been investigated using both oscillatory and
steady shear techniques12.13. Effects typical of transient
polymer networks were observed. They were inhibited by
0141-8130/83/040222-07503.00
(~) 1983 Butterworth & Co. (Publishers) Ltd
222 Int. J. Biol. Macromol., 1983, Vol 5, August
charide units) and the results of the oscillatory measure-
ments suggested that the network was transient rather
than permanent. The study was made in sodium chloride.
Experimental
Materials
Two preparations of hyaluronate were used, both gifts
of Pharmacia (Uppsala, Sweden), which will be referred to
as A and B. They were prepared from rooster comb by
methods similar to those of Swann 14. The protein content
as determined by the Folin-Ciocalteau phenol reagent is
less than 0.2~ of the organic material. An attempt to
assess the polydispersity of these materials by gel chro-
matography on Sepharose CL2B failed as they eluted in
the void volume of the column. An extensive study of the
polydispersity of similar rooster comb hyaluronate pre-
parations has been carried out using a specially prepared
0.5% crosslinked Sepharose gel 15. These studies included
light scattering and viscometric methods but were perfor-
med entirely in sodium chloride.

:a
2O
10
015 1 I0 115
Sin 2 e/2 • 30o0c
6C
rb
5C
40
....
/ ./ /-./___.-------------/" /
30 / ..... ~ /_____.~,
\
2O
1©
- - ~ ' - - - - S - ~ s _ _ :,: Z j
o'~ ~ 'o ~ '~
SiN2 0/2 +3OOOc
Figure 1 Zimm plots of light scattering data at 20°C from
hyaluronate solutions in 4 M guanidinium chloride pH 7.0. (a)
Preparation A at concentrations of 1.2,0.80, 0.60, 0.4, 0.2 and 0.1
mg ml- 1. The optical constant K = 1.78 x 10-7 ml 2 g - 2 cm-4.
(b) Preparation B at concentrations of 1.7, 1.37, 0.85, 0.64, 0.43,
0.21 mg m1-1. The optical constant K = 1.78 x 10 -7 ml 2 g-2
cm -4
Light scattering
Solvents for light scattering were made up from analyti-
cal grade reagents using distilled deionized water. The
solvents were filtered through a Buchner funnel of 1.0/~m
pore size and used directly to dissolve weighed quantities
of freeze dried preparations of hyaluronate. Concen-
trations were determined by bringing an accurately
weighed quantity of hyaluronate, corrected for water
content, into 20 ml of solvent and thereafter making the
other solutions by serial dilution into 20ml volumetric
flasks at 20°C.
To achieve Donnan equilibrium the serial dilutions
were each dialysed separately against the solvent for 12-
24 h with the pH adjusted to 6.0. Other measurements of
concentration were obtained from uronic acid determi-
nations by an automated procedure ~6. All light-scattering
cells and the wide-base pipettes used were thoroughly
water washed then cleaned in refluxing acetone and
housed in a dust-free cabinet in which the cells were
loaded.
The solutions and solvents were clarified from dust by
centrifugation. Typically the solutions were spun at
20000g for 2 - 3 h but a number of experiments were
carried out to test the effect on clarification (at higher g
forces for longer times). In general the results were not
significantly perturbed.
In some experiments a set of light scattering solutions
was manipulated further by increasing the ionic strength
and/or adding a different counterion. In such experiments
all solutions were dialysed to the new condition, the
Effect of cations on interaction of hyaluronate chains: J. K.Sheehan et al.
concentrations checked by carbazole analysis and then
reclarified by centrifugation.
The intensity of the light scattered at angles between
30 ° and 150 ° was determined with a Sofica model
4200 photogoniodiffusometer (SociOtO Franqaise
d'Instruments de ContrOle et d'Analyses, 78 Le Mesuil-
Saint-Denis, France), with a light-source of 436 nm wave-
length. Calibration of the instrument was performed at
room temperature, i.e. 21 + l°C, with redistilled Analar
benzene of Rayleigh ratio 45.6 x 10 6 c m -1, and an
additional calibration employed the polymer solid stan-
dard provided with the instrument. The data were
210 215 evaluated by the reflection correction outlined by Tomim-
atsu et al. ~7. Linear least-squares analysis yielded the
extrapolates to c = 0 and 0 = 0 , the latter values being
obtained from the data at 30 °, 34 °, 37.5 ° and 45 °. Radii of
gyration were calculated from the slope of the c = 0 line by
using the data at 30 °, 34 °, 37.5 ° and 45 °.
Refractive index-increments of the various stock
/
solutions against the appropriate buffers were determined
with a Brice-Phoenix differential refractometer at 436 nm
(Phoenix Division of Virtis Gardiner, NY 12525, USA).
U ltracentrifugation
Sedimentation rate experiments were performed in an
MSE Centriscan 75 analytical ultracentrifuge. Epoxy cells
~,o ~,~ of 1 cm path length and fitted with quartz windows were
used for all measurements. These experiments were
carried out at 54 000 rev/min, and monitored by schlieren
optics. Positions of peak maxima were used for obtaining
the sedimentation rates. So values from 1/s versus time
graphs were corrected to standard conditions of 20°C in
water.
Viscometry
The shear dependence of the viscosity was studied using
a laboratory constructed viscometer combining the
Couette and cone and plate principles TM. This allowed
viscosities of small quantities of solutions (0.92 ml) to be
determined. The detection system relied upon an optical
level on a fine wire suspension, typically 0.003 in. diameter
copper beryllium wire was used. Deflections were de-
termined using a torsion head capable of measuring to
within 1/60 ° and temperature was controlled to 0.1°C.
Readings were taken at a number of fixed shear rates at
0.5, 1, 3, 7.8 and 19 s-1 and extrapolations to zero shear
and zero concentration were made. Some experiments
were performed at fixed shear rates and this is indicated in
the text. Data were plotted as ~lsp/c,i.e. (~/T~- 1)/c, against
shear rate and the values at zero shear plotted against
concentration.
Results
Light scattering
Light scattering measurements were made with solu-
tions in a completely undisturbed state and any assoc-
iations persisting between groups of particles should be
readily observed in the weight-average molecular weight
and to a greater extent in the Z-average radius of gyration.
The possibility that residual associations were present in
this material due to the presence of non-covalently bound
protein was tested by performing experiments in 4 M
guanidinium chloride. Figure 1 shows the Zimm plots
obtained for both preparations under this condition. The
Int. J. Biol. Macromol., 1983, Vol 5, August 223
Effect of cations on interaction of hyaluronate chains." J. K.Sheehan et al.
Table I Light scattering and viscosity data from rooster comb hyaluronate

Conditions An
Sample pH 5,5~.5 10 -6 x Particle wt Radius of gyration n (nm) ~c(mlg-1) [t/](mlg -1)

A 4 M Gu HCl 3.1 128 0.10

A 0.2 M KCl 3.4 219 0.146 5000
A 0.2 M KCl 3.2 205
A 0.2 M NaC1 4.4 197
A 0.2 M NaC1 6.4 263 0.15
A 0.2 M NaCI 5.7 230 7100
A 0.066 M CaC12 10.0 310 7700
B 4 M Gu HCI 1.1 110 0.10
B 0.2 M KC1 0.95 147 0.146 3000
B 0.2 M NaC1 1.75 215 3400
B 0.066 M C a C I 2 1.7 155 3100
B 0.2 M KC1 1.1 117
B 0.2 M KCI+0.02 M NaC1 2.2 163
B 0.2 M KCI+0.1 M NaC1 2.0 153
B 0.2 M KC1 + 0.2 M NaC1 2.3 177
B 0.1 M NaC1 2.8 395
B 0.3 M NaC1 2.8 220
B 0.6 M NaC1 2.9 217

molecular weights (Table 1) were 3.1 x 106 for preparation
A and 1.1 x 10 6 for B.
Z i m m plots obtained in K +, N a + and Ca 2+ chloride
for preparation A are shown in Figure 2 and the data for
both preparations are listed in Table 1. F o r preparations A
and B the particle weight in 0.2 M KC1 coincided with
those measured in 4 M guanidinium chloride. It is likely
that these values represent the true weight average
molecular weights of these hyaluronate preparations. The
particle weights obtained in 0.2 M NaC1 and 0.066 M
CaC12 were always higher than those in 0.2 M KC1. The
values for preparation A increased from 3 x 10 6 ( K + f o r m )
to 4.4-6.4x 10 6 (Na + form); those for preparation B
increased from 1.0 x 10 6 ( K + form) to 1.7-2.7 x 10 6 (Na +
and Ca 2 +). To test whether the enhanced particle weights
were due to large microgel particles of poorly dissolved
material, the centrifugation time was varied between 1
and 3.5 h and the gravitational field between 20000 and
350009 but this had no significant effect on the result.
Increasing the ionic strength from 0.1 M NaCI to 0.6 M
NaCI also had no effect on the observed particle weight.
The effect of mixing NaC1 with KC1 is shown in Table I.
An increase in the particle weight from 1.1 x 10 6 to 2.1
X 106 occurred after the initial introduction of NaC1 at
0.02 M and this value was retained up to 0.2 M NaC1.
Ultracentrifugation
Ultracentrifugation experiments on preparation A
were made to obtain an alternative way of estimating the
molecular weight (Figure 3). In 0.2 M NaC1 and 0.066 M
CaC12 there was a departure from linearity at low
concentration and only the six lowest concentrations have
been used in the least squares analysis, yielding an S O of
12.2 x 10-13 in C a f l 2 and 11.03 x 10-13 in NaC1. In 0.2 M
KCI all the data were fitted to a straight line, giving an So
of 10.3 x 10- 13.
Viscosity
Preparation A : Figure 4 is a plot ofqsp/C against c for
three different solvent conditions at a single shear rate
(7.8 s-l). The experiments were performed on similar
solutions employed for light scattering and for which the
data were shown in Figure 2. The intrinsic viscosity in the
presence of 0.2 M NaC1 was 7100 ml g-1 while that in
0.2 M KC1 and 0.066 M CaC12 was 5000 ml g - 1. The value
for the intrinsic viscosity in the presence of CaC12
indicated a discrepancy with the light scattering data that
prompted a more detailed study of the shear dependence
of the solutions. In the presence of CaC12 all the solutions
had a non-linear shear dependence below the shear rate of
10 s-1. Figure 5a shows the shear dependence of the
viscosity at a number of concentrations while the con-
centration dependence of qsp/C(60---~0) is plotted in Figure
5b. The final value for the intrinsic viscosity is
7700 ml g - a. The effect of CaC12 on the shear dependence
and concentration dependence of the viscosity of hyal-
uronate solutions was examined further. Figure 6 shows
the effect on the relative viscosity of adding concentrated
CaCI2 to a solution of hyaluronate (0.85 mg ml-1) in 0.2 M
NaCI in the Couette viscometer. The change in relative
viscosity is achieved at a level of 20 mM CaC12. Control
experiments adding NaC1 of the same ionic strength
yielded no change in the relative viscosity.
Preparation B: Figure 7 shows rl~p/Cat 0 and 19 s - ~ as a
function of concentration in 0.2 M KC1 and 0.066 M CaC12.
In 0.2 M KC1 the shear dependence of the viscosity below a
concentration of 0.5 mg ml i was linear and small. Above
0.5 mg m1-1 the shear dependence of the viscosity
increased steadily with concentration and became more
non-linear below shear rates of 10 s-1. The intrinsic
viscosity at zero shear was 3000 ml g - 1 and 2800 ml g - 1 at
19 s - I . In 0.2 M NaC1 the data are similar at con-
centrations below 0.5 mg m l - 1 but there was an increase
in the shear dependence with concentration compared to
experiments in KC1. The qsp/C a t zero shear was 3400 ml
g - 1 while it was 3000 ml g - 1 at 19 s - 1. In 0.066 M CaC12
the intrinsic viscosity at high shear, i.e. 19 s - 1 was 1750 m l
g 1. However, as with preparation A, all concentrations
showed a non-linear increase in the shear dependence of

224 Int. J. Biol. Macromol., 1983, Vol 5, August

 Effect of cations on interaction of hyaluronate chains: J. K.Sheehan et al.

a suitable d e s c r i p t i o n for the m o l e c u l e when d a t a are

15 e x t r a p o l a t e d to zero c o n c e n t r a t i o n a n d shear rate. T h e
M a n d e l k e r n a n d S h e r a g a e q u a t i o n 19"

no[q]l/3So N
10 fl - (1 - @)(100)1/3M2/3

(where I-q] is the intrinsic viscosity, n o the solvent

/-/L 7 ./ , viscosity, S o the s e d i m e n t a t i o n rate at zero c o n c e n t r a t i o n ,
i--Y-7-7"---7
,,~_---7 - - - / " - - ; - - - ~ ' /
./ f the p a r t i a l specific volume, p the s o l u t i o n d e n s i t y a n d M
the m o l e c u l a r weight) gives a m e a s u r e of the a s y m m e t r y of
a particle in solution. M e a s u r e m e n t s by C l e l a n d 5 on
h y a l u r o n a t e fractions o f different m o l e c u l a r weights gave
i i i
05 10 15 210
Sm 2 e / 2 + 3oooc
15
0,4
b

./2"
~o ~ .... :7--7- .7" 0.3
/
./"

..... 7_W /
/ ~O. 2

J
L~_--
O.1
0~ "~ 15 20 25
%n 2912 + 3 0 0 0 c

15 c
I I I I I
01 02 03 04 0.5 06
1 0 3 x c ( g m l -~)

~c /~--- A' /.." ~ / ~ " Figure 3 Variation of sedimentation rate plotted as I/S versus
concentration for preparation A in 0.2 M NaC1 (I), 0.2 M KC1
\ (O) and 0.066 M CaC12 (A)

,~-,; "----z/ ~d 20
05 10 15 20 25
Sin 2 e l 2 + 3 0 0 0 c

Figure 2 Zimm plots of light scattering data at 20°C from

hyaluronate solutions of preparation A in: (a) 0.2 M KCI, pH 7.0.
The concentrations used were 0.52, 0.34, 0.21, 0.14 and 0.08 mg O1
m l - 1 and the optical constant K = 2.46 x 10- 7 ml 2 g - 2 cm - 4. (b)
0.2 M NaC1, pH 6.0. The concentrations used were 0.52, 0.34,
0.21, 0.14 and 0.08 mg m1-1. The optical constant
"~10
K =2.60 x 10 - 7 ml 2 g - 2 cm-4. (c) 0.066 M CaCI2, pH 6.0. The gl
concentrations used were 0.53, 0.35, 0.21,0.14 and 0.03 mg m l - 1. F"
The optical constant K=2.60 x 10 -7 ml 2 g-2 c m - 4
o
"..._

the viscosity b e l o w a s h e a r rate of 10 s-~. T h e intrinsic

viscosity qsp/C((O--~0)= 3050 ml g - *, i.e. r a t h e r similar to I I
KC1. 0.5 1.0
10 3 × c (gml 4)

Discussion Figure 4 Variation of viscosity (plotted as r/sp/C) versus con-

Average molecular shape and dimension
T h e d a t a p r e s e n t e d here are in b r o a d a g r e e m e n t with
dilute s o l u t i o n d a t a for h y a l u r o n a t e a l r e a d y p u b l i s h e d
a n d referred to. T h e m o d e l of a stiff r a n d o m coil is a
centration at a shear speed of 7.8 s - 1, and 20°C for preparation
A: I , in 0.2 M NaCl at concentrations of 0.07, 0.15, 0.2 I, 0.36 and
0.53 mg m l - l ; O, in 0.2 M KC! at concentrations of 0.05, 0.08,
0.24, 0.33, 0.47 and 0.78 mg m l - l ; A, in 0.066 M CaCI 2 at
concentrations of 0.08, 0.14, 0.31, 0.35, 0.53 and 0.87 mg m1-1

Int. J. Biol. M a c r o m o l . , 1983, Vol 5, A u g u s t 225

 Effect of cations on interaction of hyaluronate chains: J. K.Sheehan et al.

Molecular interaction
8
15 (a) (K ÷ versus N a ÷). Light scattering measurements on
both preparations show higher molecular weights in
NaCI. This may be due to (i) the presence of small
amounts of large aggregates due to poorly solubilized
material, or (ii) a general tendency of the molecules to
remain paired. The inability to get lower particle weights
Eh by increasing centrifugation time and speed supports (ii).
The resultant increase in molecular weight after dialysing
a set of light scattering solutions from KC1 into NaCI also
\ lO suggests that (ii) is a more favourable hypothesis. In
C'I
F-
tO preparation A, the higher intrinsic viscosity in 0.2 M NaC1
(7100 ml g-1 as against 5000 ml g-~ in 0.2 M KC1) is in
good agreement with the above conclusion. In prepara-
o tion B the comparative increase in viscosity from K ÷ to
...1_
N a ÷ is much less, i.e. 3000 ml g - 1 to 3400 ml g - ~ suggest-
ing that the tendency towards aggregation in NaC1 is
..I.
enhanced for larger molecules.
The concentration at which molecular domains overlap
in solution is given by a dimensionless quantity c[q]
called the coil parameter. For chains obeying the F l o r ~
Fox relationship c[q] = 1.5. For preparation A, in NaCI
I I I I
2 4 6 8 and KC1, this would predict chain overlap at a con-
S-1 centration of 0.24).3 mg ml-1. In preparation B chain
overlap occurs at a concentration around 0.5 mg m l - 1. In
b both A and B a marked increase in the concentration
15 dependence of the viscosity occurs beyond these con-
O3 centrations and for preparation B, where detailed studies
have been performed, an increase in the shear dependence
of the viscosity is seen. This increase in viscosity implies
o that hyaluronate chains in Na + and K + do not inter-
t 10
3 penetrate significantly and tend to 'bind' on touching.

Q_

Fm 5
r,3 X

1.4.
0.5
103x C ( g m l -~)
Figure 5 (a) Variation of viscosity (q~p/C) versus shear rate for
preparation A in 0.066 M CaC12 at concentrations of 0.14, 0.35,
0.53 and 0.87 mg ml- 1. (b) Concentration dependence of (qso/C)
at ~o-~0 s- 1 from data in (a) giving a final value of[q] = 7700 ml 1.3
g i
t_

ff-
an average value of 3.0 × 10 6 for this parameter. Re-
measurement of the term ~ by Wik et al. ~5 would give a
corrected value offl =2.3 × 106. Taking a mean value offl 1.2 D

= 2.6 × 10 6 and using the data for preparation A (Table 1)

gives values for the molecular weight of 3.0 x 106 in 0.2 M
KC1, 5.35 x 106 in 0.2 M NaC1 and 5.8 x 106 in 0.066 M
CaC12. These values are in reasonable agreement with
those found by light scattering. F o r random coil polymer 1.1
solutions intrinsic viscosity is related to hydrodynamic
volume by the F l o r y - F o x equation [q]=q5 63/2Ra/M, I I I I
where R is the radius of gyration, M is the molecular 20 40 60 80
weight and ~b a universal constant with a value close to 2.6 [Co 2.] in 0 . 2 M NaCI(mM)
× 1023 (Ref. 20). The average value of 4, for preparation A
is 1.8 x 1023 and B, 0.8 × 1023 which is in reasonable Figure 6 Variation of relative viscosity of preparation A at
0.85 mg m1-1 in 0.2 M NaCl at ~o=7.8 s -1 versus CaCl2
agreement with the theoretical value, given that the value concentration. Aliquots (10/A) of 2 M CaC12 were added directly
for M is a weight average and the value of R is a Z average to the hyaluronate solution in the viscometer. The new de-
and no information about polydispersity is available. flection was read after 1 min

226 Int. J. Biol. Macromol., 1983, Vol 5, August

 Effect of cations on interaction of hyaluronate chains: J. K.Sheehan et al.

71, These studies suggest that such 'binding' is more long

lived in the presence of N a ÷ than K ÷ giving a higher
6 particle weight, larger intrinsic viscosity and greater
increase of shear dependence of viscosity with con-
centration. Recent rheological studies 12 suggest that
5 interactions in sodium hyaluronate are due to specific
segmental interactions which are long lived and this is
consistent with the data discussed here.
(b) Effect of Ca2+: there was a marked effect on
solutions of hyaluronate especially in the 'dynamic'
experiments, i.e. viscosity and sedimentation rate. Light
scattering measurements gave a molecular mass greater
than that in 0.2 M KC1 but there was not a concomitant
increase in the Re. Viscosity measurements showed two
1 clear effects c o m m o n to A and B: (i) a strong shear
dependence of the viscosity between 0-10 s - 1 even for low
i concentrations of hyaluronate and (ii) a slower increase of
1.0 viscosity with concentrati6n (Figures 4 and 7). These data
suggest that in the presence of Ca 2 + (i) there is a tendency
for hyaluronate chains (under quiet conditions) to 'bind'
71b together, (ii) the internal stiffness of the hyaluronate
molecule is decreased and the chains interpenetrate more
6 readily and (iii) a combination of(i) and (ii) leads to weakl2~
stabilized aggregates of molecules disrupted by low shear
rates. Furthermore, the titration curve in Figure 6 shows
that the effects are apparent at 2 mM CaCI2 in 200 mM
NaCI.

Present solution data in relation to X-ray and n.m.r, studies

ll In potassium hyaluronate at pH 4~9, X-ray evidence
suggests an extended structure which requires consider-
× 2-
able hydration to maintain a stable environment for the
m cation, thus chain-chain interactions are mediated by
o water molecules (Sheehan and Atkins, unpublished re-
1- sults). When N a ÷ is the supporting cation the hyaluronate
molecule can coordinate an environment for the ion with
I or without supporting water molecules 7. Thus direct
1.0 hydrogen bonding between molecules is probably more
favourable in the presence of Na ÷ than K ÷ and
7 interactions between chains are more likely to persist in
¢
dilute solution. In the presence of Ca 2 ÷ the X-ray data
6- suggests that the cation is coordinated by pairs of
antiparallel chains, together with a considerable number
of supporting water molecules. The hydration of the
5- chains requires that all inter-chain bridges are made via
water molecules 9 and this would be consistent with weak
associations between molecules in dilute solution. The
increased activity of the water molecules around the
polyanion in the presence of Ca 2 ÷ might also lead to
weaker intra-chain hydrogen bonds thus making the
molecule more flexible. A number of n.m.r, studies on
hyaluronate21 - 23 are consistent with the interpretation
above. In NaCI at p H 7.0 such studies suggest that
1 hyaluronate may be viewed as a mixture of flexible and
rigid regions existing in dynamic interplay. The intro-
I
duction of Ca 2 ÷ causes an increased flexibility consistent
1.0 with the above considerations.
1 0 3 x c(gm1-1)
Figure 7 The concentration dependence of (t/s~/c) at co = 0 s- 1
(open symbols ©, [], /x) and co = 19 s-1 (closed symbols I , I , Conclusions
A) for preparation B: (a) in 0.2 M KCI at concentrations of 0.l,
0.25, 0.5, 1.0 and 1.4 mg ml-~ (©, t ) ; (b) in 0.2 M NaCI at (1) Towards infinite dilution and zero shear rate
concentrations of 0.1, 0.2, 0.4, 0.8, 1.0 and 1.4 mg m1-1 (v1, I ) ; hyaluronate adopts the conformation of an expanded
(c) in 0.066 M CaCl 2 at concentrations of 0.1, 0.25, 0.5, 0.75 and random coil in supporting electrolytes of KCI, NaCI or
1.5 mg ml-1 (A, A) CaCI2.

Int. J. Biol. Macromol., 1983, Vol 5, August 227

Effect of cations on interaction of hyaluronate chains: J. K.Sheehan et al.

(2) In NaC1 and CaC12 aggregates of hyaluronate J.K.S. was a Visiting Scientist at the University of Lund
chains persist into dilute solutions but not in KC1. (Swedish Medical Research Council-5731).
(3) Above concentrations where chains overlap
References
(C=1.5/[~/] according to statistical theory), the rapid
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228 Int. J. Biol. Macromol., 1983, Vol 5, August