Fluorescence polarization as an approach to study the molecular interaction between lupin seed γ-conglutin and insulin

Abstract

γ-Conglutin is an unusual protein found lupin in seeds. Numerous scientific data indicate that this protein is capable of reducing glucose levels in the blood. Unfortunately, the molecular mechanisms underlying this unique feature remain poorly understood, mostly due to experimental obstacles. Interestingly, γ-conglutin shows also insulin-binding ability, however, there is limited data about the kinetics of this interaction. Moreover, the available information comes from studies in which γ-conglutin was immobilized or partially unfolded. Herein, the fluorescence polarization assay application allowed for a successful determination of the insulin-γ-conglutin complex's binding parameters. Saturation and competitive binding experiments were done in a solution where γ-conglutin and insulin retained their native structure. The dissociation (Kd) and inhibition (Ki) constant of the complex was 9.16 × 10−6 M and 7.34 × 10−6 M, respectively. It is expected that knowing the quantitative interaction parameters can help in explaining the γ-conglutin hypoglycemic activity.