Conformational transition of hyaluronic acid carboxylic group participation and thermal effect

263

B}ochimica et Biophysica Acta, 541 (1978) 263--269 ~) Elsevier/North-Holland Biomedical Press

BBA 28552

C O N F O R M A T I O N A L T R A N S I T I O N OF H Y A L U R O N I C ACID CARBOXYLIC G R O U P PARTICIPATION AND T H E R M A L E F F E C T

JOON WOO PARK a and BIRESWAR C H A K R A B A R T I a,b

a Eye Research Institute o f Retina Foundation, Boston, Mass. and b Department o f Ophthalmology, Harvard Medical School, Boston, Mass. 02114 (U.S.A.) (Received November 21st, 1977)

Summary The chiroptical, viscosity and titration studies of hyaluronic acid in mixed organic/water solvent show a reversible conformational transition of the molecule depending u p o n pH, solvent composition, temperature, and molecular weight. Neither methylhyaluronate nor chondroitin undergoes conformational transition of this type: These results indicate that hydrogen bonding between the protonated carboxylic group and carbonyl oxygen of the acetamido group is directly involved in the conformational change. Results with chondroitin provide further support for the 4-fold helical structure that we have proposed for hyaluronic acid in mixed organic/water solvent. The thermal stability of the conformation has been studied under various pH values and solvent compositions.

Introduction It has become increasingly apparent that the physiological function of a biomacromolecule is closely related to its molecular conformation. Previous X-ray (ref. 1 and references therein) and chiroptical [ 2] studies have shown that hyaluronic acid can a d o p t a variety of helical conformations in a highly condensed state such as solid films and fibers. Other physicochemical investigations [ 3--7 ] have proposed a similar ordered conformation of hyaluronic acid in solution. Recently, however, some doubts have been cast u p o n the helical conformation of hyaluronate in a dilute solution at neutral pH [2,6]. In previous papers [8,9], we have demonstrated a conformational transition of hyaluronic acid from a large change in its chiroptical and hydrodynamic properties in acidic organic/water solvents. Circular dichroism (CD) showed a new positive band near 225 nm and a strong negative dichroism below 200 nm in mixed solvents, whereas in aqueous solution it exhibits a CD minimum at

264

210 nm and a maximum near 190 nm. The optical rotatory dispersion (ORD) trough at 220 nm in aqueous solution also shifts to blue with increasing m~gnitude upon addition of organic solvent. The conformational transition was observed to be cooperative with respect to pH and solvent composition. The present communication describes a detailed study of hyaluronic acid, methylhyaluronate and chondroitin, which we conducted in order to explore the nature of the transition and thermal stability of the new conformation. Materials and Methods

Sodium hyaluronate, prepared from rooster comb, was obtained from Biotrics, Inc. Chondroitin sulfate was obtained from Sigma, and was desulfated to yield condroitin according to Kantor and Schubert [10]. Methylhyaluronate was prepared by shaking finely-powdered sodium hyaluronate in methanol which contained 0.5% (v/v) acetyl chloride. Methylhaluronate was saponified to obtain hyaluronic acid. For these reactions, we followed the same work-up procedures as for the preparation of chondroitin. Standard techniques were employed for preparation of solutions. CD and ORD values were recorded in a Cary 60 and a JASCO UV/ORD-5 spectropolarimeter using a 1-cm pathlength water-jacketed cell, through which water could be circulated at constant temperature. Temperature of solutions in the cell was measured by a thermocouple; the temperature gradient inside the cell was less than 0.5°C. Variations of refractive index with solvent composition and temperature were not corrected. Viscosity was measured with a capillary viscometer in a constant temperature water bath. After optical and viscosity measurements, the solutions were brought to room temperature, and pH and hyaluronic acid concentration were determined. Titration studies were performed at room temperature and the pH values are apparent ones. Results and Discussion

The intrinsic viscosity values of both methylhyaluronate and saponified methylhyaluronate were approx. 30 cm3/g at pH 2.6 compared to 1100 cm3/g of untreated hyaluronic acid at the same pH value. These results indicate that the molecular weights of methylhyaluronate and saponified methylhyaluronate are significantly lower than that of untreated hyaluronic acid, even though the former substances are not dialyzable. CD spectra of methylhyaluronate, saponifled methylhyaluronate (low molecular weight hyaluronic acid) and hyaluronic acid in 20% ethanol/80% water at pH 2.6 are shown in Fig. 1. Hyaluronic acid shows an increased negative CD ellipticity near 230 nm due to the carboxylic group, when pH of the solution is lowered. The apparent invariant CD properties of methylhyaluronate with pH indicate that the esterification of the molecule is nearly quantitative. The CD spectrum of methylhyaluronate is identical, in shape and position, to that of hyaluronic acid in acidic aqueous solution. However, it shows substantially lower rotational strength than untreated hyalutonic acid, which could be due to the lower molecular weight of the molecule. The CD property of low molecular weight hyaluronic acid, which differs from that of methylhyaluronate, in acidic ethanol/water suggests that the carboxylic

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group is directly involved in the conformational transition of hyaluronic acid. However, the CD property change of low molecular weight hyaluronic acid is considerably smaller than that of untreated hyaluronic acid in the same solvent condition. This indicates that the stability of the new conformation of hyalutonic acid is dependent also on the molecular weight of the molecule, in addition to solvent composition, pH and temperature. A previous CD study [9] showed that the conformational transition of hyaluronic acid with decreasing pH of a solution in 20% ethanol at room temperature becomes complete when the apparent pH of the solution is approx. 3.15; the simple acid-base property of monobasic acid with the same pKa value (3.23) as hyaluronic acid predicts only 55% of protonation. If the protonated carboxylic group is indeed a participant in the conformational change, the unusual variation in pH of the solution is expected with conformational transition by titrating with a proper solution. To test this hypothesis we titrated hyaluronate in aqueous and in 20% ethanol media with acid, and titrated -hyaluronic acid and glucuronic acid aqueous solutions with 50% ethanol. The difference in behavior of the titration curves (Fig. 2), in the presence or absence of the conformational transition, provides conclusive evidence of direct involvement of the protonated carboxylic group in the conformational transition of hyaluronic acid. The variations of pH during titrations of hyaluronate in aqueous solution and of glucuronic acid can be explained by the simple acidbase equilibria of the molecules. Cooperative conformational transition of hyaluronic acid promotes protonation of the carboxylate group. Since H÷ for the protonation is provided by the solution media, the pH of the solution

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decreases slowly (Fig. 2A) or increases sharply (Fig. 2B) with conformational transition, compared to simple acid-base behavior. These titration studies also point to the critical solvent conditions for the conformational transition, such as pH ~ 3.5 in 20% ethanol, or pH 3.2 in 16% ethanol, which agrees well with previous results [ 9 ]. Temperature
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obtained from an aqueous solution shows a concave curve with a maximum near 35°C. The minimum ~sp value was consistently lower in ethanol/water solvent than in aqueous solution at the same temperature. Within the range of hyaluronic acid concentration, 0.3--0.7 mg/ml, no significant change in transition temperature was observed. The thermal transition is reversible, b u t the reverse process in rather slow. However, the original conformation is achieved by leaving the solution at approx. 4°C overnight. The dependencies of conformational transition temperature on pH and solvent composition are shown in Fig. 5. The intramolecular hydrogen bonding between carboxyl and acetamido groups as allowed in the 4-fold helical structure [1] has been proposed for the conformation of hyaluronic acid in an acidic ethanol/water solvent [8,9] at room temperature. However, the direct participation of the protonated carboxylic group in the new conformation and the apparent blue shift of the ~r-~r* amide transition with very large ellipticity [9] suggest that the hydrogen bonding is between carboxylic acid hydrogen and carboxyl oxygen of the acetamido group, rather than between the carboxylate and amide hydrogen suggested in hyaluronate films [1,2]. In both cases, ~r-~r* amide transitions were observed to be optically active in the CD spectra. Chondroitin, which is chemically similar to hyaluronic acid except in the configuration of the C-4 hydroxyl group of glucosamine moiety, did not show conformational transition in acidic ethanol/water as seen in hyaluronic acid. Low molecular weight hyaluronic acid, which, based on viscosity results, may have a molecular weight comparable to that to chondroitin, still shows spectral change in mixed solvent, whereas chondroitin does not. This result agrees well with X-ray diffraction studies [1], which proposed a hydrogen bonding between the C-4 hydroxyl group of glucosamine residue and ring oxygen of the glucuronic acid component, in addition to the carboxylate-acetamido hydrogen bond in a 4-fold helical structure of hyaluronic acid. Since the intramolecular conformational transition is accompanied b y a change in the orientation of the sugar ring with respect to the glycosidic linkage, and the energy that stabilizes the new conformation is so weak, the conformational stability can be achieved

268

only by cooperative transition. This process is likely to be "all or none". The gradual thermal transition observed, especially in low ethanol concentration or higher pH, could be due to the polydispersity of hyaluronic acid rather than the equilibrium between t w o configurations within a single molecule, which is sterically highly unfavorable. The intrinsic viscosity of hyaluronic acid in acidic ethanol/water solvent is lower than that in an acidic aqueous solution [9]: 920 cm3/g in 10% ethanol and 1150 cm3/g in an aqueous solution at pH 2.6 at 25 ° C. Such a decrease in the intrinsic viscosity of the new conformational state obtained in acidic ethanol/ water (compared to that in aqueous solution) cannot account for the increased bulk viscosity, the sharp decrease in viscosity with thermal transition, and minimum specific viscosity value in a transition region. However, this behavior with regard to viscosity can be explained in terms of interaction properties of the molecules associated with molecular conformations. Different molecular conformations give rise to different backbone structures of the molecule, resulting in different intermolecular interaction, and hence, viscosity values. This intermolecular interaction seems very strong between molecules in the new conformational state stabilized in acidic ethanol/water, b u t weak between different conformational states of the molecule. In the previous study [9], we proposed that the molecular conformation of hyaluronate in an acidic aqueous solution could be similar to the 3-fold helical model [1], and that in a dilute solution at neutral pH, the polymer is randomly coiled. However the possibility of helical conformation at neutral pH in a concentrated solution cannot be ruled out. Studies [11] suggested that hyaluronate molecules o c c u p y a large excluded volume. This effect leads to strong intermolecular association and probably to a helical conformation, indicated in the solid films [1,2]. Even in a dilute solution at neutral pH, the helical conformation similar to the 4-fold structure is possible in a proper molecular environment. Figueroa and Chakrabarti [12] demonstrated that the Cu 2÷ can chelate with both carboxyl and acetamido groups of hyaluronate, which have similar spectral and viscosity properties, as shown here. The conformational features of hyaluronic acid are thus dependent on the hydration and the interaction properties of the molecule. Considering the fact hyaluronic acid and other glycosaminoglycans are c o m m o n l y associated with protein and lipid (in membrane) containing large amounts of non-polar regions, the possibility of some ordered conformation of these biopolymers in a physiological environment is great enough to warrant further investigation. Acknowledgements This investigation was supported b y P.H.S. research grant EY-01760-01 and Research Career Development Award IK04 EY-00070 from the national Eye Institute, National Institutes of Health. Editorial assistance was provided by S. Flavia Blackwell. References 1 W i n t e r , W . T . , S m i t h , P.J,C. and A r n o t t , S. ( 1 9 7 5 ) J. Mol. Biol. 9 9 , 2 1 9 - - 2 3 5 2 B u f f i n g t o n , L . A . , P y s h , E.S., C h a k r a b a r t i , B. a n d Balazs, E.A. ( 1 9 7 7 ) J. A m . C h e m . Soc. 9 9 , 1 7 3 0 - 1734

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3 Hirano, S. and Kondo-Ikeda, S. (1974) Biopolymers 13, 1357--1366 4 ~ Chakrabarti0 B. and Balazs, E.A. (1973) J. Mol. Biol. 78, 135--141 5 Darke, A.0 Finer, E.G., Moorhouse, R. and l%ees, D.A. (1975) J. Mol. Biol. 99, 477--486 6 Mathews, M.B. and Decker, L. (1977) Biochim. Biophys. Acta 498, 259--263 7 Barrett, T.W. and Harrington, R.E. (1977) Biopolymers 16, 2 1 6 7 - - 2 1 8 8 8 Park, J.W. and Chakrabarti, B. (1977) Biopolymers 16, 2 8 0 7 - - 2 8 0 9 9 Park, J.W. and Chakrabarti, B. (1978) Biopolymers, in the press 10 Kantor, T.G. and Schubert, M. (1957) J. Am. Chem. Soc. 79, 152--153 11 Laurent, T.C. (1970) in Chemistry and Molecular Biology of the Intercellular Matrix (Bslazs, E.A., ed.), Vol. 2. pp. 703--732, Academic Press, New York 12 Figueroa, N. and Chakrabarti, B. (1978) Biopolymers, in t he press