Characterisation of separated end hyaluronan oligosaccharides from leech hyaluronidase and evaluation of angiogenesis

• 0.42 g HA4 and 0.46 g HA6 were obtained from 1 g HA with 16,000 U/mL LHase.
• Accurate control of the proportion of HA4 and HA6 was achieved.
• Highly pure HA4 and HA6 were prepared by ion exchange Q-Sepharose Fast Flow.
• HA4 and HA6 from LHase showed high activity in promotion of angiogenesis.

Hyaluronan oligosaccharides (o-HAs), especially saturated o-HAs, have attracted intensive attention due to their potential applications in medical treatments. In this study, the hydrolysis process of leech hyaluronidase (LHase) towards the hyaluronan was investigated by HPLC and HPLC/ESI–MS. The proportions of hyaluronan tetrasaccharide (HA4) with hexasaccharide (HA6), end products, were illustrated to have a relationship with the amount of LHase. Higher yield of HA4 was achieved with higher activity of LHase. After optimisation of the packing resin and operation parameters (balanced pH, elution concentration, elution volume and elution flow rate), the highly pure HA4 and HA6 were efficiently separated and prepared by combining ion exchange Q-Sepharose Fast Flow and size exclusion column chromatography. Compared with o-HAs (average Mr of 4000 Da), HA4 and HA6 were demonstrated to show higher activity for promoting angiogenesis, which was similar with the corresponding HA4 and HA6 produced by bovine testicular hyaluronidase. The pure HA4 and HA6 that prepared from LHase will attract intensive studies and be used in potential applications in near future.