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Purification and characterisation of gliadin fractions from wheat flour

Yeboah, Nana Asare (1991) Purification and characterisation of gliadin fractions from wheat flour. Doctor of Philosophy (PhD) thesis, University of Kent. (doi:10.22024/UniKent/01.02.86087) (KAR id:86087)

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https://doi.org/10.22024/UniKent/01.02.86087

Abstract

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

Item Type: Thesis (Doctor of Philosophy (PhD))
DOI/Identification number: 10.22024/UniKent/01.02.86087
Additional information: This thesis has been digitised by EThOS, the British Library digitisation service, for purposes of preservation and dissemination. It was uploaded to KAR on 09 February 2021 in order to hold its content and record within University of Kent systems. It is available Open Access using a Creative Commons Attribution, Non-commercial, No Derivatives (https://creativecommons.org/licenses/by-nc-nd/4.0/) licence so that the thesis and its author, can benefit from opportunities for increased readership and citation. This was done in line with University of Kent policies (https://www.kent.ac.uk/is/strategy/docs/Kent%20Open%20Access%20policy.pdf). If you feel that your rights are compromised by open access to this thesis, or if you would like more information about its availability, please contact us at ResearchSupport@kent.ac.uk and we will seriously consider your claim under the terms of our Take-Down Policy (https://www.kent.ac.uk/is/regulations/library/kar-take-down-policy.html).
Uncontrolled keywords: Biochemistry
Subjects: Q Science > QH Natural history > QH426 Genetics
Q Science > QK Botany
Divisions: Divisions > Division of Natural Sciences > Biosciences
SWORD Depositor: SWORD Copy
Depositing User: SWORD Copy
Date Deposited: 29 Oct 2019 16:28 UTC
Last Modified: 14 Feb 2022 12:14 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/86087 (The current URI for this page, for reference purposes)
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