High-performance capillary electrophoresis separation of hyaluronan oligosaccharides produced by Streptomyces hyalurolyticus hyaluronate lyase

Abstract

The action of Streptomyces hyalurolyticus hyaluronate lyase on hyaluronic acid (HA) determined by high-performance capillary electrophoresis (HPCE) (electrokinetic chromatography with sodium dodecyl sulphate) was examined and compared with the HPLC procedure. By using an uncoated fused-silica capillary tube, 50 μm ID, 65 cm long from the injection point to the detector, the separation of HA species from DP 4 to approx. DP 30 was obtained. The length of the capillary was found to be fundamental for the separation and resolution of the unsaturated HA oligosaccharides. Furthermore, the HPCE separation of HA Δ-tetrasaccharide and Δ-hexasaccharide species produced a greater detection sensitivity (about 20 times greater) than HPLC. Various HA samples were analyzed for the ratio of tetrasaccharide/hexasaccharide ( T/ H) after treatment with S. hyalurolyticus hyaluronidase. HA samples of extractive origin of different molecular masses showed a T/ H ratio between 1.28 and 1.48 determined by HPCE, with a good correspondence with the HPLC separation. On the contrary, a sample of cross-linked HA resulted in a great discrepancy between the two analytical techniques (greater than 40%). HA of fermentative origin had a T/ H ratio of approx. 1.92–2.00, close to that of HA (approx. 1.80) for the first time extracted and purified from the body of a species of mollusc bivalve, Mytilus galloprovincialis. The presence of resistant (less susceptible to enzyme cleavage) site repeating unit in the carbohydrate backbone of HA is discussed in relation with the T/ H values experimentally determined by HPCE.