Electron microscopic visualization of the polysaccharide hyaluronic acid.

A method for making mucopolysaccharide molecules visible in the electron microscope is reported and is applied to elucidate the structure of hyaluronic acid. The method is essentially that of Kleinschmidt and Zahn' 2 for nucleic acids: the polyanion strands are combined with a positively charged protein, such as cytochrome c, incorporated into a surface film of the protein, and then shadowed with electron dense material. Individual strands can be seen, and therefore molecular structure may be investigated. This was not possible in earlier attempts to visualize hyaluronic acid in the electron microscope,3-6 in which, as Gross3 4showed, undefinable though possibly fibrillar aggregates were observed.7 Hyaluronic acid is of structural importance in animal connective tissue,8 in the vitreous body of the eye,9 and is the lubricating component of joint fluid.10 Physical evidence indicates that hyaluronic acid molecules behave in solution as relatively stiff chains.7' 8, 11-14 The solutions show considerable particle-particle interaction,7' 14-17 and this led Preston, Davies, and Ogston14 to raise the question whether hyaluronic is a straightor branched-chain molecule. Chemical evidence18 shows that it is a linear polymer of N-acetylglucosamine and glucuronic acid, but a few branch points in a polymer of several thousand hexose units could elude detection1 and yet radically alter the appearance of the molecule and its interactions. To determine whether the chains are linear or branched, hyaluronic acid molecules were spread on a surface and examined in the electron microscope; a distribution of chain lengths was also obtained.