Activation of Limulus Coagulation Factor G by Scleroglucan as a Biological Activity Measurement

Abstract

The ability of scleroglucan (beta-1,3-beta-1,6 glucan) conformers to activate Limulus coagulation factor G was evaluated. Studies pursued to get a further insight into the scleroglucan structure-function relationship. Lab-scale produced (EPS I, EPS II, EPSi) and commercial (LSCL) scleroglucans were tested on their ability to activate factor G of the Limulus coagulation cascade (Glucatell Test, Pyrolab). Native triplex conformation was obtained by dissolving scleroglucan (2 pg/mL) in distilled water. Thermally- (150°C, 30 min) and alkali-treated (0·2 N NaOH, 10 min) scleroglucans were also prepared. Single helices were further evaluated at 2 ng/mL and 2 µg/mL. All scleroglucans were endotoxin-free (LAL-Test, Pyrolab). Triplex conformation had stronger ability to activate factor G than single helix, with EPS I and EPS II as the most active polymers. Thermal and alkaline denaturation significantly reduced scleroglucan reactivity, being EPS II the most stable glucan, whilst EPSi and LSCL were the most affected ones (91-99% reduction). Single helix activation ability was significantly dependent on polysaccharide concentration. Scleroglucans from S. rolfsii ATCC 201126 (EPS I and EPS II) exhibited marked clotting activity against factor G and were more effective than commercial scleroglucan. Denaturation would lead to lower biological activity and it would be dependent on EPS concentration.