SODIUM HYDROXIDE-INDUCED CONFORMATIONAL CHANGE IN SCHIZOPHYLLAN DETECTED BY THE FLUORESCENCE DYE, ANILINE BLUE

Molecular conformation is considered to be an important factor in dete rmining the biological activity of glucans; however, a simple method t o detect the conformation change for glucans in solution has not been developed. We found that the fluorescence intensity of aniline blue bo und 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 moni tor conformational change that is simpler and easier to perform than o ther techniques (such as solid-state C-13 NMR spectroscopy). The nativ e conformation for SPG [a branched beta-(1-->3) glucan] is a rigid, cl osed triple helix. Treatment with NaOH, followed by neutralization, pr oduces a single helix-rich preparation. We observed that aniline blue does not stain native SPG, but will stain the renatured NaOH-treated S PG. This suggests that aniline blue binds only to single helix forms o f SPG. Further supporting evidence is that the fluorescence intensity is decreased on consecutive days after neutralization, which is consis tent 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. Ake tagawa, 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 reactivi ty of renatured SPG, stabilized with aniline blue at different days, w as evaluated using a glucan sensitive LAL. The activity of LAL toward SPG was decreased over time, suggesting that the conformation of gluca n detected by fluorescence intensity correlated with the LAL activity. (C) 1998 Elsevier Science Ltd. All rights reserved.