Application of limulus test (G pathway) for the detection of different conformers of (1-->3)-beta-D-glucans.

Abstract

The reactivity of factor G mediated coagulation pathway in limulus amebocyte lysate which is triggered by (1-->3)-beta-D-glucans is thought to depend on the structure of the glucans, especially on the ultrastructure: triple helix, single helix and random coil. We used Sonifilan (SPG) and grifolan (GRN) as parent compounds to compare the reactivities of these three conformers. Under a neutral condition, alkaline treated SPG (SPG-OH, single helix) and polycarboxylated SPG (PC-SPG, random coil) showed significantly stronger reactivity than untreated SPG (triple helix). After the alkaline treatment, all three conformers showed comparable reactivities. It is suggested that the pretreatment of the glucan preparations by sodium hydroxide is quite important to compare quantitatively the reactivity of the glucans by limulus test, and comparing the data of untreated and alkaline treated glucans would provide information about their conformations. Using this approach, it was found that after heat treatment at around 150 degrees C, the conformation of GRN was changed to rich in the triple helix, and that following sodium hydroxide treatment and dialysis of GRN, the conformation of GRN was changed to single helix rich conformer. About half of the single helix conformer was gradually changed to triple helix conformer over one week at 4 degrees C.