259

Biochimica et Biophysica Acta, 498 (1977) 259--263 © Elsevier/North-Holland Biomedical Press

BBA Report BBA 21452

CONFORMATION OF HYALURONATE IN NEUTRAL AND ALKALINE SOLUTIONS

MARTIN B. MATHEWS and LaVERNE DECKER

Departments of Pediatrics and of Biochemistry, The University of Chicago, Chicago, Ill. 60637 (U.S.A.) (Received January 26th, 1977)

Summary Increasing the pH of a neutral salt solution of sodium hyaluronate to 12.5 produces a rapid drop in viscosity which is reversible upon restoring the pH to neutrality. Light scattering data showing a decrease in radius of gyration with no change in molecular weight and negative results with chondroitin and other acidic glycosaminoglycans suggest that the conformational change is specific for hyaluronate molecules.

Previous studies by several investigators have shown that glycosaminoglycans can adopt a variety of ordered conformations in such highly condensed states as exist in oriented films and fibers. However, evidence for the existence of ordered glycosaminoglycan conformations under conditions of high hydration in the physiological environment was obtained only for hyaluronate [1]. In particular, recent chiroptical measurements by Chakrabarti and Balazs [2] and Hirano and Kondo-Ikeda [ 3 ], and nuclear magnetic relaxation data by Darke et al. [4] confirm earlier conclusions [1] that the conformation of the polymer is more random at high pH than at neutral pH. The present work explores the relationship between molecular structure and size and the conformational change produced in hyaluronate by an increase in pH. Glycosaminoglycans were prepared and characterized by previously described methods [5]. Chondroitin 4-sulfate and chondroitin 6-sulfate were chemically desulfated to yield chondroitin [6]. Bacterial hyaluronate was purified by anion exchange resin chromatography [5] of a preparation from Streptococcus pyogenes. Hyaluronate fractions (GF26C and GF26F) were prepared by treatment of umbilical cord hyaluronate with testicular hyaluronidase (EC 4.2.2.1; purchased from Leo-Helsinborg laboratories, Sweden) and fractionation on a column of Sephadex G-200.

260

Viscosity average molecular weights (Mv) were estimated from measured values of intrinsic viscosity and empirically established relationships between intrinsic viscosity and molecular weight [5]. The light scattering methods used for determination of the molecular parameters of hyaluronate are described elsewhere [ 7 ]. Fig. 1 shows in a typical fashion the rapid drop in reduced viscosity of hyaluronate solutions produced by raising the pH to 12.5 and the equally rapid reversal of this parameter upon returning the pH to near neutrality. However, prolonged exposure to pH 12.5 results in depolymerization of hyaluronate (verified by isolation of products and determination of My), although, without affecting the property of reversible change in reduced viscosity with pH. The initial effect of increased pH is to produce a change of macromolecular conformation without depolymerization. This conclusion is supported by light scattering data that show a change of less than 2% in the molecular weight of one million concomitant with a drop in the root mean ~quare radius of gyration from 112 nm to 94 nm. No change in reduced viscosity upon increase of pH from 5.5 to 12.5 was found for the following glycosaminoglycans of indicated viscosity average molecular weights: chondroitin 6-sulfate (3.0-104), chondroitin 4-sulfate (1.2-104), chondroitin (1.0. 104), and dermatan sulfate (2.7-104). The molecular weight of chondroitin determined by sedimentation equilibrium was 0.98.104 . Table I shows that pH values of 10.7 or less have little or no effect on the reduced viscosity of sodium hyaluronates from different biological sources. Also, the effect of increasing pH to 12.5 on hyaluronate fractions from human umbilical cord are very similar for preparations in the molecular weight range 2000q

1600

-6

o

1200

I i I I I I

0 800 NEUTRALIZED

1'

400

NEUTRALIZED ..... 0

t

I

I

i

I

[~

I

1

2

3

4

5

6

20

HOURS F i g . 1. E f f e c t o f p H u p o n r e d u c e d v i s c o s i t y o f u m b i l i c a l c o r d h y a l u r o n a t e ( M v = 1 - 1 0 6 ) . I n t h e first e x p e r i m e n t , t h e average f l o w t i m e o f 1 . 0 0 m l o f a s o l u t i o n o f h y a l u r o n a t e in 0 . 4 M N a C ] , p H a b o u t 5 . 5 , w a s d e t e r m i n e d i n a u b b e I o h d e , s u s p e n d e d o l e v e l v i s e o m e t e r at 2 5 . 0 0 ° C, A t t = 0 , 0 . 0 2 m l o f 2 . 5 M N a O H w a s a d d e d , t h e s o l u t i o n m i x e d a n d t h e average f l o w t i m e at p H 1 2 . 5 d e t e r m i n e d . A t t ~ 8 r a i n , 0 . 0 2 m l o f 3 . 0 M a c e t i c acid w a s a d d e d a n d t h e a v e r a g e f l o w t i m e at p H 5 . 5 d e t e r m i n e d . In t h e s e c o n d e x p e r i m e n t , 0 . 0 2 m l o f 2 . 5 M N a O H w a s a d d e d t o 1 . 0 0 m l o f h y a l u r o n a t e s o l u t i o n in 0 . 4 M N a C I , t h e s o l u t i o n m i x e d , a n d t h e a v e r a g e f l o w t i m e d e t e r m i n e d at t = 0 a n d at v a r i o u s t i m e intervals t h e r e a f t e r u p t o 2 0 h . A t this t i m e t h e s o l u t i o n w a s n e u t r a l i z e d b y t h e a d d i t i o n o f 0 . 0 2 m l 3 . 0 M a c e t i c a c i d a n d t h e average f l o w t i m e d e t e r m i n e d . S o l u t i o n s w e r e e x p o s e d t o a t m o s p h e r i c o x y g e n at all t i m e s .

261 of 13--100-104 and only somewhat smaller for preparations of about 1- 104 molecular weight. The effect of increasing pH to 12.5 on hyaluronate was not significantly influenced by the presence of 6 M urea. The light scattering data showing a decrease in radius of gyration of the hyaluronate molecule with no change in molecular weight upon increase in pH confirms previous suggestions [2,3,4] that the reversible molecular transition is one from a more-extended to a less extended form of a single chain molecule. The conformational change has also been interpreted as an orderdisorder transition. An important question relates to the chemical change produced by high pH. One possible effect of alkali is the ionization of hydroxyl groups, perhaps the C-4 hydroxyl groups of glucosamine residues, with resulting destabilization of the ordered structure. The pH dependence shown in Table I is suggestive of hydroxyl group ionization with a pK about 12. High pH ionization of hydroxyl groups has been invoked to explain a comparable molecular transformation shown by amylose [8]. Darke et al. [4] concluded that the hyaluronate molecule at neutral pH contained both "flexible" chain segments and "stiff" chain segments and that treatment with 1 M NaOH converted most of the latter segments reversibly to a more flexible form. They also suggested that the "stiff" chain segments, which are capable of adopting a relatively ordered conformation differ from "flexible" chain segments by some minor but as yet uncharacterized covalent features, and have a minimum size of 60 disaccharide units. Our viscosity data, however, are inconsistent with these conclusions based upon nuclear magnetic resonance observations. A significant, and possibly constant, degree of "stiffness" or ordered structure persists in hyaluronate even after reduction of the chain size from 2500 to 23 disaccharide units. Data indicating comparable molecular conformational transitions with increase of pH have been obtained now for hyaluronates from human umbilical cord and rooster comb by nuclear magnetic resonance methods [4] and for hyaluronates from rooster comb [9], umbilical cord, rabbit skin, and Streptococcus pyogenes by viscometry. It is possible that the macromoTABLE

I

EFFECT OF pH ON PERCENT OF VARIOUS SOURCES AND

REDUCTION MOLECULAR

OF REDUCED WEIGHTS

VISCOSITY

OF SODIUM

HYALURONATES

Sample I n t r i n s i c viscosity ( m l / g ) : 10 -4 X Mv:

F235E* 1800 100

F253C* 580 24

pH 9.5 p H 10.7 pH 11.8 pH 12.5

0 4 27 44

. -. 50

F254C* 350 13 .

GF26C** 58 1.3

.

. 2

-.

. 46

GF26F** 44 0.9

RHA-I*** 100 2.7

HAS-1 t 280 10

--

0 21 45

2 21 44

.

. 36

37

* F r o m h u m a n umbilical cord. * * F r o m h y d r o l y s i s o f F 2 3 5 E b y t e s t i c u l a r h y a l u r o n i d a s e ; values o f M v are a p p r o x i m a t e . M o l e c u l a r w e i g h t s b y s e d i m e n t a t i o n e q u i l i b r i u m for G F 2 6 C and G F 2 6 F are 1 . 0 7 . 1 0 4 a n d 0 . 9 3 o104, r e s p e c t i v e l y , w i t h v e r y n a r r o w ranges o f m o l e c u l a r w e i g h t d i s t r i b u t i o n for e a c h preparation. * * ; F r o m r a b b i t skin. From Streptococcus pyogenes.

262 lecular chains of these hyaluronates are similarly constituted of the proposed "stiff" segments and "flexible" chain segments distributed in block polymer fashion. It should be noted, however, that the observed constancy of conformational properties would require the presence of two different chemical types of segments in remarkably similar proportions in hyaluronates from widely different sources. In contrast to hyaluronate, all other known kinds of glycosaminoglycans of connective tissues vary in proportions of chemical types of disaccharide repeating units distributed within the same molecular chain or contained in preparations from different sources. These sources may be tissues from the same animal, homologous tissues of the same species at different stages of development, or homologous tissues of different species [1]. Hypothetical models for the extended, ordered state of hyaluronate in dilute aqueous solution at neutral pH, are 3-fold and 4-fold helical forms deduced from X-ray fiber diffraction data [10,11]. Both structures are stabilized in part b y a set of systematic intrachain H-bonds between the C-4 oxygen atom of a glucosamine residue and the ring oxygen of a glucuronate residue. The absence of a high pH effect on the reduced viscosity of chondroitin suggests that the configuration of C-4 of the hexosamine residue (the sole difference between disaccharide repeats of hyaluronate and chondroitin) may be crucial to stabilization of a helical conformation. A biological specificity for the C-4 configuration, and possibly for ordered conformation also, is indicated by the ineffectiveness of chondroitin in competing with hyaluronate for binding to cartilage proteoglycan molecules [12]. Evidence for a helical conformation of hyaluronate in solution rests primarily upon previous chiroptical data [2,3] and also upon observations of a reversible circular dichroic transition occurring between 75 to 92 ° [13]. However, some doubts have been cast recently upon the origin of the chiroptical effect [14]. In addition, spin-spin relaxation processes in hyaluronate solutions appear to be t o o fast to arise from a stable, rigid conformation [4]. Furthermore, we note that a denaturant such as urea in 6 M concentration affects neither the drop in viscosity increment of hyaluronate at high pH nor the proportion of "stiff" and "flexible" segments at neutral pH [4]. Thus, a helical conformation for the ordered state of hyaluronate at neutral pH is a problematical issue. Streptococcal hyaluronate was a gift from Dr. H. Album. Dr. T.C. Laurent very kindly determined the molecular weights of t w o samples of hyaluronate and of a sample of chondroitin by sedimentation equilibrium. This work has been supported by a grant-in-aid from the American Heart Association with funds contributed in part by the Chicago and Illinois Heart Association and by a grant from the National Institutes of Health, HD-04583. We are greatly indebted for helpful criticism to Drs. T.C. Laurent, and W.T. Winter, and particularly to Dr. D.A. Rees for valuable comments and a copy of an unpublished manuscript. References 1 M a t h e w s , M.B. ( 1 9 7 5 ) C o n n e c t i v e Tissue: M a c r o m o l e c u l a r S t r u c t u r e a n d E v o l u t i o n , pp. 2 1 2 - - 2 2 3 , Springer-Verlag, Berlin

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2 3 4 5 6 7 8 9 10 11 12 13 14

Chakxabazti, B. and Balazs, E.A. (1975) Fed. Proc. 34, 635 Hizano, S. and Kondo-Ikeda, S. (1974) Biopolymers 13, 1357--1366 Darke, A., Finer, E.G., Moorhouse, R. and Rees, D.A. (1975) J. Mol. Biol. 99, 477--486 R o d i n , L., Baker, J.R., Cifonelli, J.A. and Mathews, M.B. (1972) in Methods in E n z y m o l o g y (Ginsbttrg, V., ed.), Vol. 28, pt. B, pp. 73--140, Academic Press, New Y ork Kantor, T.G. and Schubert, M. (1957) J. Am. Chem. Soc. 7 9 , 1 5 2 - - 1 5 3 Mathewe~ M.B. and Lozaityte, I. (1958) Arch. Biochem. Biophys. 74, 158--174 Rao, V.S.R. and Foster, J. (1963) Biopolymers 1 , 5 2 7 - - 5 4 4 Swann, D.A. (1970) in Chemistry and Molecular Biology of the Intercellular Matrix (Balazs, E.A., ed.), Vol. 2, pp. 734--748, Academic Press, New York Winter, W.T., Smith, P.J.C. and Arnott, S. (1975) J. Mol. Biol. 99, 219--235 Guss, J.M., Hukins, D.W.L., Smith, P.J.C., Winter, W.T., Arnott, S., Moorhouse, R. and Rees, D.A. (1975) J. Mol. Biol. 95, 359--384 Hascall, V.C. and Heinegard, D. (1974) J. Biol. Chem. 249, 4242--4249 Scott, J.E. (1976) Front. Matrix Biol. 3, 176--191 Balazs, E.A., McKinnon, A.A., Morris, E.R., Rees, D.A. and Welsh, E.J. (1977) Chem. Commun., in the press