Perspective Experimental approaches to hyaluronan structure Mary K. Cowman a, * and Shiro Matsuoka b a Othmer Department of Chemical and Biological Sciences and Engineering, Polytechnic University, 6 Metrotech Center, Brooklyn, NY 11201, USA b Department of Biomedical Engineering, Columbia University, 503 West 120th Street, MC8904, New York, NY 10027, USA Received 16 October 2004; accepted 10 January 2005 Dedicated to Professor David A. Brant, in honor of his pioneering work on carbohydrate conformation Abstract—A review of the literature describing experimental studies on hyaluronan (HA) is presented. Methods sensitive to the hydrodynamic properties of HA, analyzed in neutral aqueous solution containing NaCl at physiological concentration, can be shown to fit the expected behavior of a high molecular weight linear semi-flexible polymer. The significant nonideality of HA solu- tions can be predicted by a simple treatment for hydrodynamic interactions between polymer chains. Nuclear magnetic resonance and circular dichroism studies of HA are also in agreement with a model incorporating dynamically formed and broken hydrogen bonds, contributing to the semi-flexibility of the polymer chain, and segmental motions on the nanosecond time scale. HA shows the capability for self-association in the formation of a viscoelastic putty state at pH 2.5 in the presence of salt, and a gel state at pH 2.5 in mixed organic/aqueous solution containing salt. Ordered and associated structures have also been observed for HA on the surfaces, especially in the presence of surface-structured water. These phenomena can be understood in terms of coun- terion-mediated polyelectrolyte interactions. The possibility that hyaluronan exists in vivo in environments that induce ordered structures and assemblies is discussed. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Hyaluronan; Polysaccharide; Viscosity; Polymer solution; Osmotic pressure; Atomic force microscopy 1. Introduction Hyaluronan (HA) is a high molecular weight linear polysaccharide, with the structure poly[(1!3)-2-acetam- ido-2-deoxy-b-D-glucopyranosyl-(1!4)-b-D-glucopyr- anosyluronic acid]. It has one carboxylate group per disaccharide repeat, and is therefore a polyelectrolyte. There are no known deviations from the linear repeating disaccharide structure, with the possible exception of occasional deacetylated glucosamine residues. The molecular weight of HA can range as high as 6000– 8000 kDa, in synovial fluid, eye vitreous, and rooster comb. 1 A stretched chain of HA with a molecular weight of6000 kDawouldhaveanapproximatelengthof15 lm, and a diameter of about 0.5 nm. Isolation of HA from normal skin, muscle, heart, lung, small intestine, large intestine, etc., frequently yields a lower molecular weight polymer, but this is probably the result of degradation during isolation, since recent data for HA isolated from those tissues in the presence of desferoxamine shows only high molecular weight. 2 HA can be chemically degraded by hydroxyl radicals and peroxynitrite, 3 which may be generated in inflammatory conditions or during tissue remodeling. For example, reduced molecular weight HA has been observed in osteoarthritis. 4,5 HA is found primarily in the extracellular matrix and pericellular matrix, but has also been shown to occur intracellularly. The biological functions of HA include maintenance of the elastoviscosity of liquid connective tissues such as joint synovial fluid and eye vitreous, con- trol of tissue hydration and water transport, supramo- lecular assembly of proteoglycans in the extracellular matrix, and numerous receptor-mediated roles in cell 0008-6215/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2005.01.022 * Corresponding author. Tel.: +1 718 260 3054; fax: +1 718 260 3125; e-mail: mcowman@poly.edu Carbohydrate RESEARCH Carbohydrate Research 340 (2005) 791–809