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

摘要

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 mum 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 A-tetrasaccharide and A-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 TIH values experimentally determined by HPCE.