Quasi-elastic light scattering studies of hyaluronic acid solutions

648

Biochimica et Biophysica A cta, 343 (1974) 648--655

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 27390 QUASI-ELASTIC LIGHT SCATTERING STUDIES OF H Y A L U R O N I C ACID SOLUTIONS

F. ROSS HALLETT and A. LAURENCE GRAY Department of Physics, University of Guelph, Guelph, Ontario (Canada)

(Received November 9th, 1973)

Summary Autocorrelation functions for the intensity of laser light scattered from solutions of hyaluronic acid have been measured under a variety of experimental conditions. The form of these functions is consistent with the large amount of long-range intra- and intermolecular interactions characteristic of polyelectrolyte solutions. Further experiments have been performed in order to study the effect of hyaluronic acid on the Brownian diffusion of polystyrene spheres. Using several different sphere sizes as solution probes, the importance of the ionic environment in determining hyaluronic conformation has been demonstrated.

Introduction Hyaluronic acid is a linear mucopolysaccharide which plays an important role in determining the physical properties of synovial fluid, vitreous humour, and several other b o d y fluids. The physical and chemical properties of hyaluronic acid have been reviewed by Laurent [1] and b y Blumberg and Ogston [2]. The molecular weight and conformation in solution has been studied b y light scattering [3--6], viscosity [3,6,7], sedimentation [5,6], streaming birefringence [8], and gel electrophoresis [10,11]. Because of the polyelectrolyte nature of hyaluronic acid the molecular domain is very large and straightforward interpretation of the results of many of these experiments is possible only when experiments are conducted at high dilutions. A further complication in these experiments arises because samples are polydisperse, the average degree of polymerization depending on the source and method of preparation of the sample [11]. Recent X-ray crystallographic studies [12,13], have supported the theory that the repeat unit in hyaluronic acid is a disaccharide composed of N-acetylglucosamine and glucuronic acid. Films of hyaluronic acid prepared from gels at pH 2.5, where the glucuronic acid moiety is not dissociated, have recently

649 been shown to contain long helices which are presumably stabilized b y hydrogen bonds in a manner quite similar to the a-helices found in proteins. However, at physiological pH values the glucuronic acid is dissociated (pK = 3.21) [1], the polymer is heavily charged, and another configuration, probably an extended random coil, is present. The present work is an a t t e m p t to learn more about these interactions and the way in which t h e y determine the properties of the solution. Quasi-elastic light scattering has been used in this study because of its demonstrated ability to follow molecular dynamics in solution. While pure hyaluronic acid solutions have been investigated, most of the experiments discussed below are measurements of the translational diffusion exhibited b y polystyrene spheres in the solutions. The specific advantage of the light scattering m e t h o d is its ability to measure the effective viscosities experienced b y different sphere sizes when the solution is at rest. This eliminates the need for extrapolation of results to zero shear conditions. A further advantage of the technique is that one is measuring the effective viscosity experienced b y the spheres. For polymeric solutions this "microscopic" viscosity can differ considerably from the bulk viscosity and provide us with new information a b o u t the microscopic environment of the solution. Theory and Experimental Methods Quasi-elastic light scattering [14,15], is becoming a widely used tool in the study of the motion of scattering particles in solution. The various techniques used in analyzing quasi-elastic light scattering experiments have been applied to the study of biopolymers [ 1 6 ] , polymers [17], bacteria [18], virus [ 1 9 ] , and cells [20]. I n the case of Brownian motion of independent spherical scatterers the experimental result of this light scattering technique is normally a normalized intensity autocorrelation function, CI(T) which can be interpreted simply in terms of the translational diffusion coefficient of the scattering particle. The function C I (T) has the form C~(v) = 1 + f(c) exp (--2DK:T)

(1)

where f(c) is related to the coherence of the scattered light and is a constant for a particular experimental set-up, D is the translational diffusion coefficient of the scattering particles, T is time, and K is the scattering vector given b y K =

4~ 0 ~- sin

(2)

In Eqn (2) ~ is the wavelength of the light in the solvent and 0 is the scattering angle. The intensity correlation function Cx (T) is related to the magnitude of the electric field correlation function of the scattered light b y the equation. LCE (T)I = ~/CI (T) -- 1 = f ' ( c ) e - D K 2 r

(3)

The diffusion coefficient D, is the only unknown parameter in Eqn (3) and it is routinely obtained b y least squares computer fit to the experimental autocorrelation function.

650 For the case mentioned above the translational diffusion coefficient of the spherical particles is related to the particle radius " a " through the Stokes-Einstein equation kT

D -

(4)

67r~?a

where k = Boltzmann's constant, T is absolute temperature and ~ is the viscosit y of the solution. Defining the so-called correlation time re as rc -

1

(5)

DK 2

then the specific viscosity experienced by a monodisperse sample of spherical particles in a solution is Tc'

r~sp

-

re"

DO -- 1 = ----I

D

(6)

In this expression rc' and D are the correlation time and diffusion coefficient of the polystyrene spheres in the solution; %,, and Do are the corresponding properties for spheres in the pure solvent under identical conditions. The scattered laser light is detected with a cooled RCA C31024A photomultiplier and correlation of the amplified, discriminated pulse train is performed by an on-line PDP-9 computer. The autocorrelation function is generated digitally in a manner already described [21], but using a much improved computer interface. This interface is composed of two 20-bit registers driven by a 10-MHz clock together with four temporary 18-bit storage buffers. The device measures time intervals between pulses in units of 200 ns and transfers these intervals sequentially in batches of 4000 to the computer memory. The dead time of the system is 800 ns. With this system it is possible to perform full autocorrelation analysis on data collected at count rates as high as 25000/s with negligible effects due to the small 800-ns dead time. The intensity autocorrelation function is produced digitally in as m a n y as 128 channels of a Nuclear Data Storage and Display oscilloscope. The background in this function is calculated for each batch and the sum of these numbers is used as the final background in this experiment. This minimizes the error in this background which would arise due to changes in the average scattered light level if the background was calculated at the end of this experiment. The hyaluronic acid samples were obtained as a filamentous powder from Nutritional Biochemical Corporation and from Schwartz Mann (umbilical cord*}. These samples often contained as much as 16% by weight absorbed water which could be safely removed by freeze--drying or by gentle heating in a vacuum connected to a cold trap. After the water was removed, sample vials were stored in a desiccator maintained at --20 ° C. Further, the samples from the manufacturers contained variable amounts of salt which had to be removed

*

Preparation data will be supplied on request.

651

before physical measurements were reliable and repeatable. This was accompanied by successive washings of dissolved samples in Amicon Corp CF 50A membrane filter cones which were spun at 1000 X g in a Sorvall RC-2 centrifuge. Less than 1% of the hyaluronic acid was found in the washings from the concentrate as determined by the borated carbazole assay of Gregory [22]. Results and Discussions A semi-log plot of the magnitude of the electric field autocorrelation function, ICE (T)I, obtained from a 0.1% solution of hyaluronic acid is shown in Fig. 1. The sizable departure from a straight line could be interpreted as indicating extreme polydispersity since scatterers having a range in size lead to an electric field autocorrelation function which involves a sum of exponentials. However, the correlation time obtained b y fitting the tail of the function corresponds to particles having an effective Stokes radius of order 10 #m. The molecular weight of such a particle would be many orders of magnitude greater than the typical molecular weight of hyaluronic acid (--~ 106). For this reason, we feel that the deviation from linearity in the semi-log plot is not due to polydispersity, b u t rather to extensive intermolecular interaction and possibly contributions due to intramolecular motion. The longest correlation time c o m p o n e n t disappears upon addition of NaC1 to the solution, a result which is also demonstrated in Fig. 1. The addition of salt screens the electrostatic interactions b e t w e e n the carboxyl groups o f the glucuronic acid moieties and thus significantly reduces b o t h intramolecular and intermolecular interactions. Experiments at lower hyaluronic acid concentration were attempted because lowering concentration should reduce the intermolecular effects, b u t leave intramolecular effects unchanged. However, the low light levels made the results inconclusive. Because of the difficulties encountered in analysis of the light scattered from the hyaluronic acid macromolecules, experiments have been performed using polystyrene spheres of various sizes as probes of the solution. While these 10 09 08 07

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Fig. 1. Semi-log plots o f the electric field a u t o c o r r e l a t i o n f u n c t i o n JCE(7)[ f r o m 0.1% hyaiuronic acid s o l u t i o n s c o n t a i n i n g (A) 0.000 M NaCI0 (B) 0.001 M NaCI and (C) 0.005 M NaCI.

652

20000

1500O Tc [Ms)

i

10000

B

T. ±

O

001 o52

T ±-0%3 o ~

i

/

I

oog oi

i

o~

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plotted

as a

experiments have been performed under conditions where > 95% of the scattered laser light originated from the spheres, the sphere concentrations needed to maintain this criterion were still very small relative to the solute concentration. Under these conditions it is possible to use quasi-elastic light scattering to monitor the Brownian motion of the spheres. Fig. 2 shows the variation in correlation time of the light scattered from the spheres as a function of NaC1 concentration. Only a small dependence of sphere correlation times on salt concentration has been observed in the absence of hyaluronic acid so that the large effects in Fig. 2 must arise from change in the intra- and intermolecular interactions brought about by changes in the ionic environment. The effective viscosity of the solution, and its dependence on NaC1 concentrations, as experienced by the spheres, is shown in Fig. 2. Curve A is the result of light scattering experiments using 1830 A diameter polystyrene spheres as probes. Curve B is the result when 930 h diameter spheres were used. The viscosity drops rapidly as the NaC1 concentration is increased, becoming insensitive to increases in free ion concentration above about 0.05 M. At this concentration the intramolecular and intermolecular interactions of the hyaluronic acid molecules are apparently screened by the formation of counter-ion layers. One of the most interesting features in Fig. 2, is the observation that the 930 A diameter spheres exhibit correlation times which yield lower values for viscosity than the 1830 A spheres. The effect is more pronounced at the lower

653

NaC1 concentrations. There appears to be a sieve effect in solutions of hyaluronic acid in that the small spheres are less hindered in their translational diffusion than the larger ones. This sieve effect was first observed by Laurent [23,24], who noticed that during centrifugation small particles settled through hyaluronic acid solutions much faster than larger ones. More recently, the effect on translational diffusion has been studied by Ogston et al. [25]. These experiments demonstrate that the microscopic viscosities of solutions may in certain cases be considerably different from the bulk viscosities. The observations further support the theory that the gel-like properties of hyaluronic acid solutions or synovial fluid are due to extensive inter- and intramolecular interactions and not due to copious amounts of bound water. The dependence of the reduced microscopic viscosity

T~red

sp

=

-

C

(DolD)-- I

(7)

C

as measured by the spheres on hyaluronic acid concentration is shown in Figs 3 and 4. Again the sieve effect is found in that the reduced viscosity as measured by the small spheres is less than that measured by the large spheres. The reason for the maxima in the curves appears to be related to the expansion of the hyaluronic acid molecule with dilution [26]. Dilution provides an increase in volume for the counter-ions and consequently a reduced shielding of the negative charges fixed on the polyelectrolyte. The additional coulomb repulsion leads to expansion of the molecule. It is interesting to note that the small

09 f os

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06

i

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01 0

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08

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Fig. 3. R e d u c e d specific v i s c o s i t y as m e a s u r e d b y 9 3 0 A d i a m e t e r p o l y s t y r e n e spheres and p l o t t e d as a f u n c t i o n o f h y a l u r o n i c acid c o n c e n t r a t i o n ; (A) 0.00 M NaCI0 (B) 0.05 M NaCI.

654 11 A

10 . . . . . . .°. . . . . ...... ...I . . . ...... / .... i" ......

0.9 0.8

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.............................

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! .....

0.2 0.1

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Hyaluronic

ocJd

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(~.6 0,6 0'7 3 g/cc) (xlO-

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Fig. 4. R e d u c e d specific viscosity as m e a s u r e d b y 2 4 6 0 A diameter p o l y s t y r e n e spheres and p l o t t e d as a f u n c t i o n of h y a l u r o n i c acid c o n c e n t r a t i o n ; (A) 0 . 0 0 M NaC1, (B) 0 . 0 5 M NaC1.

spheres are significantly more sensitive to this effect than the larger ones. This may be because they are less entangled and better able to probe the microscopic environment of the solution. At concentrations less than 0.1 mg/ml the individual molecules appear to be fully expanded and are beginning to separate from each other as their concentration is further reduced. This dilution leads to a reduction in the extent of intermolecular interactions and the reduced viscosity is lowered. Throughout this discussion we have taken the phenomenological approach to the translational diffusion of the polystyrene spheres. The Stokes--Einstein equation has been invoked to define a reduced specific "microviscosity" (Eqn 7). Recently Ogston et al. [25] have argued that a stochastic theory is preferable. In their treatment the concept of an effective microviscosity is avoided and the motion of the spheres is discussed in terms of hindered diffusion. If this latter approach is taken, the ordinate of Figs 3 and 4 should be left as (Do/D-1)/c rather than making the further extension to ~red/C. Acknowledgements The authors are grateful to Mrs J. Marsh for technical assistance and to Dr George Renninger for many helpful discussions. This work was supported by the Canadian Arthritis and Rheum. Society, Grant No. 51497 and by a National Research Council of Canada Grant No. 57636. References 1 Laurent, T.C. ( 1 9 7 0 ) in Chemistry and Molecular B i o l o g y of the Intercellar Matrix (Balazs, E . A . , ed.), VoL 2, pp. 7 0 3 - - 7 3 2 , A c a d e m i c Press, L o n d o n 2 Blumberg, B.S. and Ogston, A.G. ( 1 9 5 8 ) Ciba F o u n d a t i o n S y m p o s i u m on Chemistry and Biology of M u c o p o l y s a c c h a r i d e s , pp. 2 2 - - 4 1 M. Little-Brown, B o s t o n 3 Balazs, E.A., Watson, D., Duff, I.F. and R o s e m a n , S. ( 1 9 6 7 ) Arthritis R h e u m . 10, 3 5 7 - - 3 7 6 4 Laurent, T.C. and Gergely, J. ( 1 9 5 5 ) J. Biol. Chem. 2 1 2 , 3 2 5 - - - 3 3 3 5 Lau~ent, T.C., R y a n , M. and Pietruszkiewicz, A. ( 1 9 6 0 ) Biochim. Biophys. A c t a , 42, 4 7 6 - - 4 8 5

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