# An investigation of Glycine max oleosins, a Glycine max vacuole protein, and the Manduca sexta apolipophorin-III as primary emulsifiers

#### Abstract

Emulsification of oils by protein is of particular interest due to the finding that specific proteins naturally function as colloid stabilizers. The research presented focuses on the surface and interfacial properties of protein isolated and recombinantly synthesized from Glycine max, (soybean) and the Manduca sexta (tobacco hornworm), respectively. Plant proteins include a class of proteins termed oleosins and a 34kD-vacuole soy protein, both of which associate with micellar particles (oil bodies) that store and transport fats within the plant. The Manduca sexta Apolipophorin III is also of interest because of its natural association with lipophorins, or emulsion droplets, (analogous to oil bodies) within insect hemolymph. The results show that the plant and insect proteins are responsive to pH and concentration. Surface properties evaluated include equilibrium surface and interfacial tensions, dynamic surface tensions, surface hydrophobicities, and emulsion stabilities. The maximum tetradecane emulsified at short stability times was determined utilizing experimental design to assess the relative importance of the independent variables (pH and protein concentration). The highest tetradecane amounts were emulsified for both insect and plant protein and lower amounts are emulsified by the commercially used $\beta$-casein. Interfacial tensions and surface hydrophobicities agreed with emulsification data, and this may serve as predictive experiments for emulsification ability. To address the interaction of Manduca sexta Apolipophorin-III with hydrophobic substances, a mutant containing a cysteine residue in the place of a former leucine residue (at a reported docking site) was evaluated. Comparisons were made between the wild type protein, the cysteine mutant, and a resulting dimer in terms of equilibrium interfacial tensions, dynamic surface tensions. The results showed that the wild type and mutant monomer possess similar interfacial properties, whereas the mutant dimer is slightly less interfacially active. This suggested that the proposed site of binding contributed only partially to hydrophobic binding. Experimental results indicate that the proposed site is involved, but in conjunction with other non-specific hydrophobic interactions (or another site-specific anchoring region of the molecule) to interactions at hydrophobic interfaces.