Purification and characterisation of gliadin fractions from wheat flour

Three gliadin fractions designated yni-gliadin, yv-gliadin and o-gliadin were purified to homogeneity from milled flour (variety Chinese Spring) in good yield. The proteins were characterised in terms of amino acid compositions and N-terminal amino acid sequences; these confirmed that they were typical gliadins. Mr values by SDS-PAGE and by gel filtration in GuHCl, were 4IK for the yin-gliadin by both methods, 47K and 44K respectively for the yv-gliadin and 76K and 90K for the a>-gliadin. The chemical characterisation was extended using the physical techniques of fluorescence and circular dichroism spectroscopy to derive information on the proteins’ conformational properties, and using time-resolved fluorescence to probe dynamic properties. Studies in the presence of various concentrations of urea were carried out in order to monitor unfolding of the native conformation, and the proteins’ stabilities to unfolding. The wavelength of maximum fluorescence emission (approximately 350nm) indicated that the tryptophan residues of the proteins were exposed to solvent in the native state; this was confirmed by the high values obtained for the Stern-Volmer quenching constants (12-18 M'1), indicating considerable accessibility to exogenous quencher. Time-resolved fluorescence studies indicated rotational correlation times for the tryptophan side-chains of 3-6ns indicative of free mobility of these side-chains. The far-uv CD spectra of the proteins indicated some regular secondary structure; the spectra were suggestive of some a-helices in the y-gliadins and some 6-turns in the o-gliadin. Exposure of the proteins to high concentrations of urea led to some loss of far-uv CD spectral features indicating secondary structure, implying that the proteins became 4 unfolded to some extent. However, these conditions produced only small changes in fluorescence intensity and wavelength of maximum emission and led to minimal changes in the near-uv CD spectra. These results indicate that there is little change in the environments of aromatic residues on unfolding, consistent with the interpretation that these residues are exposed in the native state. Preliminary conformational and denaturation analyses were also carried out using two peptides derived from a related y-gliadin fraction corresponding to its repetitive prolinerich N-terminal and non-repetitive S-rich C-terminal domains respectively. The properties of these peptides indicated that the properties of the intact y-gliadins could be regarded as the sums of those of the isolated domain