Investigation of surface hydrophobicities of purified gliadins by hydrophobic interaction chromatography, reversed-phase high performance liquid chromatography and apolar ligand binding

The surface hydrophobicities of purified wheat α-, β-, γ- and ω-gliadins were investigated by hydrophobic interaction chromatography (HIC) on Octyl- and Phenyl-Sepharose CL-4B, reversed-phase high performance liquid chromatography (RP-HPLC) on octadecylsilane (TSK ODS 120 T) and apolar ligand (2- p -toluidinylnaphthalene-6-sulfonate, TNS) binding. Chromatographic methods, especially RP-HPLC, are tapid and easy to use, but they have some important limitations in that protein conformational structures during separation are unknown. Ligand binding is more time consuming but provides more fundamental information about protein hydrophobicity in solution and permits control of protein structure. Nevertheless, the results obtained using the three methods were in general agreement. On the basis of chromatographic experiments, a hydrophobicity scale for gliadins was established. These experiments also demonstrated a preference of gliadins for an aromatic ligand; this was particularly marked for ω-gliadins, suggesting that the surface hydrophobicities of α-, β-, and γ-gliadins depend on both aromatic and aliphatic amino acid side chains, whereas that of ω-gliadins depends mainly on aromatic side chains. The binding of TNS by gliadin molecules was co-operative and resulted in protein aggregation and masking of hydrophobic sites at high ligand/protein ratios. Gliadins differed in terms of the number of binding sites, with values ranging from 11 (β-gliadins) to 30 to 40 (ω-28): the binding constants also varied, being about ten times lower for ω-gliadins than for α-, β- and γ-gliadins.