Hyaluronan hydrolysis

Asteriou, T., Gouley, F., Deschrevel, B., and Vincent, J. C. (2002). In Influence of Substrate and Enzyme Concentrations on Hyaluronan Hydrolysis Kinetics Catalyzed by Hyaluronidase. J. F. Hyaluronan, G. O. Kennedy, and P. A. Phillips Williams (Eds.). Woodhead Publishing, Wrexham, Wales, vol. 1, pp. 249-252. 

Asteriou, T., Vincent, J. C., Tranchepain, F., and Deschrevel, B. (2006). Inhibition of hyaluronan hydrolysis catalysed by hyaluronidase at high substrate eoncentration and low ionie strength. Matrix Biology 25, 166-174. 

Asteriou, T., Deschrevel, B., Gouley F., Vincent, J.C. (2002) Influence of substrate and enzyme concentration on hyaluronan hydrolysis kinetics catalyzed by hyaluronidase, in Hyaluronan Proceedings of an International Meeting, September 2000, North East Wales Institute, UK (eds J.F. Kennedy, G.O. Phillips, P.A. Williams, V.C. Hascall), Woodhead Publishing Ltd, Cambridge, pp. 249-252. 

Tommeraas, K., Melander, C. (2008) Kinetics of hyaluronan hydrolysis in acidic solution at various pH values. Biomacromolecules, 9, 1535-1540. 

The typical biopolymers used for the preparation of micro- and nanogels are polysaccharides (Scheme 11) cellulose (CL), chitosan (CS), hyaluronan (HA), heparin, pullulan (PuL), dextran, and gelatin - a proteinaceous polyamolytic gel obtained by partial hydrolysis of collagen. Gelatine microgels are addressed by Landfester and Musyanovych in another chapter of this issue [5] and will thus not be discussed further here. 

Glycosaminoglycans are solubilized from stromal or other tissues by extracting the source tissue with dilute acid or alkali. Hyaluronan is electrostatically bound to specific proteins called hyaladherins, which possess a structural domain of -100 amino acids termed a link module. Other glycosaminoglycans are O-linked to serine and threonine residues of polypeptides and these bonds hydrolyze before the rest of the polysaccharide. The protein moiety precipitates when trichloroacetic acid or ammonium sulfate is added to the cooled mixture. The composition of the GAGs (including hyaluronan) was identified by chromatographic separation of the purified polysaccharides, followed by their hydrolysis in boiling 1.0 M HC1 for 2 1 h and identification of the individual monosaccharide components. 

It is known that the hydrolysis reaction proceeds via an oxazolinium ion intermediate. Based on this Kobayashi and coworkers succeeded in designing a disaccharide oxazoline transition state analog self-condensing substrate to revert the native action of HAase towards synthesis of hyaluronan and derivatives thereof (see Figure 9.15). 

The proposed mechanism for enzymatic hydrolysis of hyaluronan involves nucleophilic attack of water at the anomeric carbon of the d-G1cNAc moiety via an oxazolinium ion intermediate (see Figure 9.16b). A water molecule can nucle-ophilically attack the oxazolinium anomeric carbon atom to open the oxazolinium ring, resulting in the formation of the hydrolysis products of a shortened HA molecule (see Figure 9.16c). 

In the short period between 1948 and 1951, several chemists initiated research to elucidate the structure of hyaluronic acid. In 1948 A. Dorfinan published the first results of a kinetics of fermentative hydrolysis of hyaluronan [7]. Three years later in 1951, A.G. Ogston and J.E. Stanier published the first significant data about the structure of the HA macromolecule in aqueous solution. They found that the relationship between viscosity... 

The first method is a relatively simple and short process [5] that involves multiple water extractions of grounded rooster combs, precipitation from combined extracts of HA with acetic acid, treatment with base, enzymatic hydrolysis of the protein, ultrafiltration, ethanol precipitation and final dissolution of the precipitate hyaluronan in water. 

Then a solution of papain (200ml of 0.1% solution of papain per 1kg of material) is added. Enzymatic hydrolysis of proteins is carried out for 20-40h. Every 6-8 h the pH must be adjusted to 6-7 (to optimize enzyme activity). The HA mixture is purified by ultrafiltration and precipitation with 96% ethanol. The final product, sodium hyaluronate, is dried by lyophilization and the precipitate is then dissolved in a water-alcohol mixture containing 40% ethanol. The final purification of hyaluronan is performed by membrane filtration of a solution containing 30-50% ethanol. 

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