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

Abstract: Justification: Soybean agglutinin (SBA) is a carbohydrate-binding protein that is digestive enzyme resistant but heat-labile. Native SBA is tetrameric, containing four monomeric subunits. Despite SBA’s antinutritional activity being triggered by intact carbohydrate-binding sites, systematic research examining the structure-dependent bioactivity, hemagglutinating activity, and immunoreactivity of SBA is lacking.

Objectives: (1) Investigate the effect of temperature on the structure-function relationship of pH-induced SBA. (2) Predict the potential SBA structure changes during treatment using two monoclonal antibodies (mAbs) with different properties. (3) Evaluate the thermostability of pH-stressed SBA.

Methods: pH-stressed (pHs 3.0, 7.2, 8.5, and 9.6) SBA physical properties (particle size, ζ-potential, and aggregation temperature) and secondary structure were investigated using dynamic light scattering and circular dichroism, respectively. Gel electrophoresis was used to study heat-induced structural configuration changes in SBA. The effect of pH and temperature on the SBA immunoreactivity was analyzed using immunoassays. The hemagglutinating activity of heated SBA was measured by hemagglutination assay.

Results: SBA had the least thermostability at pH 3.0 and the highest at pH 8.5. Native SBA, a tetramer, caused hemagglutination. When SBA was heated, the content of antiparallel β-sheets decreased, reflecting aggregation or dissociation, which led to hemagglutinating activity reduction. Both temperature and sodium dodecyl sulfate (SDS)-induced structural changes in pH-stressed SBA altered immunoreactivity. Regardless of changes in SBA structural configuration, it was immunodetectable by anti-SBA mAb5A5, which has a thermostable linear epitope. As for another anti-SBA mAb5G12, only SBA tetramer was immunodetectable.

Significance: To reduce the antinutritional properties of SBA, one has to destroy the SBA’s tetrameric structure by applying food processing methods (e.g., heating) or adding food additives (e.g., SDS). In addition, two anti-SBA mAbs could be used to surveil food safety and toxicology, i.e., identifying the uncooked or undercooked soy products and, more importantly, quantifying antinutritional SBA in foods by targeting its conformational epitope.