Sodium hydroxide-induced conformational change in schizophyllan detected by the fluorescence dye, aniline blue

Abstract

Molecular conformation is considered to be an important factor in determining the biological activity of glucans; however, a simple method to detect the conformation change for glucans in solution has not been developed. We found that the fluorescence intensity of aniline blue bound to schizophyllan (SPG) can be used to estimate the relative amount of single helix converting to triple helix during different stages of a denature–renature cycle. This observation provides a method to monitor conformational change that is simpler and easier to perform than other techniques (such as solid-state 13C NMR spectroscopy). The native conformation for SPG [a branched β-(1→3) glucan] is a rigid, closed triple helix. Treatment with NaOH, followed by neutralization, produces a single helix-rich preparation. We observed that aniline blue does not stain native SPG, but will stain the renatured NaOH-treated SPG. This suggests that aniline blue binds only to single helix forms of SPG. Further supporting evidence is that the fluorescence intensity is decreased on consecutive days after neutralization, which is consistent with the report that NaOH-treated SPG gradually lost 77% of their single helix component in 1 week (N. Nagi, N. Ohno, Y. Adachi, J. Aketagawa, H. Tamura, Y. Shibata, S. Tanaka, and T. Yadomae, Biol. Pharm. Bull., 16 (1993) 822–828). The single helix is the conformation which activates the Limulus amebocyte lysate (LAL). The biological reactivity of renatured SPG, stabilized with aniline blue at different days, was evaluated using a glucan sensitive LAL. The activity of LAL toward SPG was decreased over time, suggesting that the conformation of glucan detected by fluorescence intensity correlated with the LAL activity.