Effect of temperature on structural configuration and immunoreactivity of pH-stressed soybean (Glycine max) agglutinin

Abstract

Soybean agglutinin (SBA) was purified using ammonium sulfate precipitation and liquid chromatography. Purified SBA was used to produce monoclonal antibodies through hybridoma technology. SBA secondary structure was studied using circular dichroism. pH-stressed (pHs 3.0, 7.2, 8.5, and 9.6) SBA physical properties (particle size, ζ-potential, and aggregation temperature) were investigated. Gel electrophoresis (non-native and native) was used to study heat-induced structural configuration changes in SBA. The effect of pH and temperature on the immunoreactivity of SBA was analyzed using enzyme-linked immunosorbent assay and immunoblots probed with two anti-SBA monoclonal antibodies with either linear or conformational epitopes. The hemagglutinating activity of heated SBA was measured by hemagglutination assay. Our results indicated that SBA had the least thermostability at pH 3.0 and the highest at pH 8.5. Temperature-induced structural configuration change on pH-stressed SBA led to immunoreactivity change. Heat-induced (70 and 80 °C) soluble SBA aggregation was proportionally related to hemagglutinating activity reduction.

Keywords: Hemagglutinating activity; Immunoreactivity; Soybean agglutinin; Structural configuration; Temperature; pH-stressed.