ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 237, No. 1, February 15, pp. 253-260, 1985 Cationic Dye Binding by Hyaluronate Fragments: Dependence on Hyaluronate Chain Length’ RAYMOND E. TURNER AND MARY K. COWMAN2 Department of Chemistry, Polytechnic Institute of New York, SSS Jay Street, Brooklyn, New York 11201 Received June 26,1984, and in revised form October 17, 1984 Sodium hyaluronate, digested with bovine testicular hyaluronidase, yielded a mixture of oligosaccharides with identical repeating disaccharide structures and differing molecular weights. The oligosaccharides were separated into a ladder-like series of bands by electrophoresis on a 10% polyacrylamide gel matrix. Coelectrophoresis of purified oligosaccharides has established that adjacent bands differ in chain length by one disaccharide unit. This procedure formed the basis for a rapid screening method in which the binding of cationic dyes by hyaluronate oligosaccharides may be assayed. As a function of chain length, the oligosaccharides showed a marked change in dye binding. Species containing less than seven repeating disaccharide units are not detected by any dye tested, even at very high sample loads. Larger oligosaccharides show an increase in dye binding. The chain length at which constant maximal dye binding is reached depends on the dye structure and solvent conditions, varying from approximately 12 to 30 disaccharide units. The hyaluronate fragments of sufficient chain length to duplicate polymer behavior should be useful models for the study of hyaluronate structure and interactions in solution. o 19% Academic press, k. Hyaluronic acid (HA)3 is a high-molec- ular-weight connective tissue glycosami- noglycan which is composed of alternating N-acetyl-@-D-glucosamine and b-D-&&W uranic acid residues linked at the 1,3 and 1, 4 positions, respectively (1). Its overall conformation in aqueous solution has been characterized as a random coil with some stiffness (2, 3). The stiffness results in part from limited conformational freedom about the glycosidic linkages (4-7). Inter- residue hydrogen bonds have also been proposed (8-13), but hydrogen bonding to solvent H20 may predominate for the amide proton (14). Rigidity may also be conferred by intramolecular or intermo- i This investigation was supported by NIH Grant EY 04804 and a Biomedical Research Support Grant to the Polytechnic Institute of New York. ’ To whom correspondence should be addressed. 3 Abbreviations used: HA, hyaluronic acid, GlcNAc, N-acetylglucosamine. lecular association of chain segments. Ev- idence for such association is derived pri- marily from rheological studies, and the capacity of polymeric HA to form a putty- like state at low pH, and a rigid gel at low pH in ethanol-H20 mixtures (15-18). Chain-chain association, leading to the formation of a network-like matrix, may be an important aspect of HA function. For example, an HA network has been proposed to support and stabilize the col- lagen microarchitecture of the human eye vitreous (19). One approach to the analysis of HA conformation and self-association is to study the molecular weight dependence of physical properties. This approach is based on the concept that a minimum number of repeating units may be required for cooperative stabilization of ordered struc- tures. It has been noted that HA frag- ments, studied in Hz0 at neutral pH, show molecular weight-dependent circular 253 0003-9861/85 $3.00 Copyright 0 1985 by Academic Press. Inc. All rights of reproduction in any form reserved.