Effect of the cations sodium, potassium and calcium on the interaction of hyaluronate chains: a light scattering and viscometric study

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 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 rheological 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

medium-sized oligosaccharide fragments ( ~ 6 0 disaccharide units) and the results of the oscillatory measurements 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 chromatography 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 preparations has been carried out using a specially prepared 0.5% crosslinked Sepharose gel 15. These studies included light scattering and viscometric methods but were performed entirely in sodium chloride.

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

:a

2O

10

015

1 I0

115

210

215

Sin 2 e/2 • 30o0c 6C

rb

5C

40

30 \

....

/ /

./ .....

/

/-./___.-------------/" / ~ /_____.~,

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 MesuilSaint-Denis, France), with a light-source of 436 nm wavelength. 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 standard provided with the instrument. The data were evaluated by the reflection correction outlined by Tomimatsu 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).

2O

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- - ~ ' - - - - S - ~ s _ _ :,:

o'~

Z

~ 'o

j

~ '~

~,o

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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 analytical 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. Concentrations 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 1224 h with the pH adjusted to 6.0. Other measurements of concentration were obtained from uronic acid determinations 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

Sedimentation rate experiments were performed in an MSE Centriscan 75 analytical ultracentrifuge. Epoxy cells 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 determined 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 solutions in a completely undisturbed state and any associations 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

Sample

Conditions pH 5,5~.5

10 -6 x

A A A A A A A

4 M Gu HCl 0.2 M KCl 0.2 M KCl 0.2 M NaC1 0.2 M NaC1 0.2 M NaCI 0.066 M CaC12

3.1 3.4 3.2 4.4 6.4 5.7 10.0

B

4 M Gu HCI

B B B

0.2 M KC1 0.2 M NaC1 0.066 M C a C I 2

B

B B B B B B

Particle wt

~c(mlg-1)

[t/](mlg -1)

128 219 205 197 263 230 310

0.10 0.146

5000

1.1 0.95 1.75 1.7

110 147 215 155

0.10 0.146

0.2 M KC1

1.1

0.2 M KCI+0.02 M NaC1 0.2 M KCI+0.1 M NaC1 0.2 M KC1 + 0.2 M NaC1

2.2 2.0 2.3

117 163 153 177

0.1 M NaC1 0.3 M NaC1 0.6 M NaC1

2.8 2.8 2.9

395 220 217

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 of

qsp/C against c for three different solvent conditions at a single shear rate 224

An

Radius of gyration n (nm)

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

0.15 7100 7700 3000 3400 3100

(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 concentration 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 hyaluronate 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 concentrations 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

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

15

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 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"

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

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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)

A' /.." ~ / ~ "

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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 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 K =2.60 x 10 - 7 ml 2 g - 2 cm-4. (c) 0.066 M CaCI2, pH 6.0. The concentrations used were 0.53, 0.35, 0.21,0.14 and 0.03 mg m l - 1. The optical constant K=2.60 x 10 -7 ml 2 g-2 c m - 4

O1

"~10 gl F"

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 KC1.

I

10 3 × c (gml

Discussion 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

I

0.5

1.0 4)

Figure 4 Variation of viscosity (plotted as r/sp/C) versus concentration 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. 8 15

Eh

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Molecular interaction (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 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 suggests that (ii) is a more favourable hypothesis. In 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 preparation B the comparative increase in viscosity from K ÷ to N a ÷ is much less, i.e. 3000 ml g - 1 to 3400 ml g - ~ suggesting that the tendency towards aggregation in NaC1 is 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 and KC1, this would predict chain overlap at a concentration of 0.24).3 mg ml-1. In preparation B chain overlap occurs at a concentration around 0.5 mg m l - 1. In both A and B a marked increase in the concentration dependence of the viscosity occurs beyond these concentrations 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 that hyaluronate chains in Na + and K + do not interpenetrate 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 g i

1.3 t_

ff-

an average value of 3.0 × 10 6 for this parameter. Remeasurement of the term ~ by Wik et al. ~5 would give a corrected value offl =2.3 × 106. Taking a mean value offl = 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 solutions intrinsic viscosity is related to hydrodynamic volume by the F l o r y - F o x equation [q]=q5 63/2Ra/M, where R is the radius of gyration, M is the molecular weight and ~b a universal constant with a value close to 2.6 × 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 agreement with the theoretical value, given that the value for M is a weight average and the value of R is a Z average and no information about polydispersity is available.

226

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

1.2

D

1.1

I

I

I

20 40 60 [Co 2.] in 0 . 2 M NaCI(mM)

I

80

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 concentration. Aliquots (10/A) of 2 M CaC12 were added directly to the hyaluronate solution in the viscometer. The new deflection was read after 1 min

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

71, 6 5

1 i 1.0

71b 6

ll

×

m o

21I 1.0 7

¢

65-

1 I

1.0 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 , 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 concentrations of 0.1, 0.2, 0.4, 0.8, 1.0 and 1.4 mg m1-1 (v1, I ) ; (c) in 0.066 M CaCl 2 at concentrations of 0.1, 0.25, 0.5, 0.75 and 1.5 mg ml-1 (A, A)

These studies suggest that such 'binding' is more long lived in the presence of N a ÷ than K ÷ giving a higher particle weight, larger intrinsic viscosity and greater increase of shear dependence of viscosity with concentration. Recent rheological studies 12 suggest that 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 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 concentrations of hyaluronate and (ii) a slower increase of 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' together, (ii) the internal stiffness of the hyaluronate molecule is decreased and the chains interpenetrate more 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 In potassium hyaluronate at pH 4~9, X-ray evidence suggests an extended structure which requires considerable hydration to maintain a stable environment for the cation, thus chain-chain interactions are mediated by water molecules (Sheehan and Atkins, unpublished results). When N a ÷ is the supporting cation the hyaluronate molecule can coordinate an environment for the ion with or without supporting water molecules 7. Thus direct hydrogen bonding between molecules is probably more favourable in the presence of Na ÷ than K ÷ and interactions between chains are more likely to persist in dilute solution. In the presence of Ca 2 ÷ the X-ray data suggests that the cation is coordinated by pairs of antiparallel chains, together with a considerable number of supporting water molecules. The hydration of the 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 hyaluronate may be viewed as a mixture of flexible and rigid regions existing in dynamic interplay. The introduction of Ca 2 ÷ causes an increased flexibility consistent with the above considerations.

Conclusions

(1) Towards infinite dilution and zero shear rate hyaluronate adopts the conformation of an expanded random coil in supporting electrolytes of KCI, NaCI or 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 chains persist into dilute solutions but not in KC1. (3) Above concentrations where chains overlap (C=1.5/[~/] according to statistical theory), the rapid increase in viscosity and shear dependence of the viscosity for hyaluronate solutions suggests that chains tend to 'bind' at contact rather than interpenetrate. (4) In the presence of Ca 2 + aggregates of chains are weakly stabilized in dilute solution and are disrupted at low shear rates, i.e. 0-10 s- 1. The presence of Ca 2 + at low levels (5-20 mM in 0.2 M NaC1) is responsible for a decrease in the concentration dependence of the viscosity which would be consistent with a more flexible chain configuration. Recent studies on hyaluronate suggest that the internal stiffness of the molecule arises from semi-cooperative intra-chain hydrogen bonding. Rheological studies suggest that the properties of hyaluronate networks arise from specific interaction between such stiff segments rather than by generalized entanglement. The studies presented here are in agreement with such conclusions and would add that segmental interactions are significantly modified by the nature of the supporting cations particularly Ca 2+. How such changes in the microenvironment of the network effect the exclusion and diffusion of other particles in it, has yet to be explored.

J.K.S. was a Visiting Scientist at the University of Lund (Swedish Medical Research Council-5731). References 1 2

3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19

Acknowledgements

20

We thank the Wellcome Foundation and the Medical Research Council for support, Department of Chemistry, Lancaster University for use of a differential refractometer and Drs I. A. Nieduszynski and Lars-Ake Fransson for invaluable discussion. While this study was completed

21

228

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