Susceptibility of Lupin γ-Conglutin, the Plasma Glucose-Lowering Protein of Lupin Seeds, to Proteolytic Enzymes

Lupin seed γ-conglutin, orally administered to animal models, has been shown to display glucosecontrolling properties. Therefore, we have addressed the study of γ-conglutin susceptibility to proteolytic enzymes in vitro as the basis to unveil its metabolic fate in the body. Pepsin treatment at pH 2.0 and 3.0 caused extensive proteolytic breakdown, while at pH 4.0, where pepsin is
minimally active, γ-conglutin was unaffected. Aliquots of the pepsin-treated protein were further incubated with pancreatin at neutral pH. If the protein backbone was already cleaved by pepsin
action, then the breakdown by pancreatin was almost complete; alternatively, pancreatin did not
affect at all γ-conglutin polypeptide chain. This was not due to an inhibitory activity of γ-conglutin,
because co-incubation with casein showed complete breakdown of the milk protein. Furthermore,
γ-conglutin was incubated with bromelain, a proteinase effective between pH 4.0 and 7.0. A sharp
transition from the uncleavable to the fully cleavable form of γ-conglutin was observed below pH
4.25. Therefore, it was concluded that (i) γ-conglutin is resistant to proteolysis at pH greater than
4.0, likely because of a compact native conformation, (ii) an acidic pH renders the protein
susceptible to proteases, suggesting the occurrence of a trans conformation, which has also been
observed by circular dichroism spectral analysis, and (iii) the protein undergoes an “all or none”
degradation pathway, regardless of the enzyme used.