Effect of calcium on structure and function of a hyaluronic acid matrix: Carbon-13 nuclear magnetic resonance analysis and the diffusional behavior of small solutes

Abstract

Natural-abundance of 13C NMR at 25.16 MHz was used to study a 2.5% matrix of hyaluronic acid [human umbilical cord] at various degrees of polymerization and at various ionic strengths. Peak assignment is facilitated by comparing proton-decoupled and off-resonance-decoupled spectra of a hyaluronidase-depolymerized matrix with spectra from relevant monosaccharides. In contrast to the spectrum following depolymerization, the spectrum for intact matrix has considerable broadening, particularly for peaks assigned to the N-acetylglucosamine moiety. This is most dramatic for the hydroxymethylene carbon. With the addition of Ca2+ above 5 mM these broadened peaks narrow and approach the sharpness observed for the hyaluronidase digest. There is no shift in resonance peak positions. These changes are quantitatively less impressive if Na+ is substituted for Ca2+. The existence of a considerable degree of order in regions of the matrix at physiological concentrations of Ca2+ is suggested. Within such a matrix the translational movement of lysine and glucose is enhanced relative to that in a matrix of agarose. Further addition of Ca2+ abrogates not only matrix order, but the enhanced diffusivity as well.