Glycoproteins have diverse and essential roles within biological systems. They are formed by enzymatic addition of saccharides to proteins during, or shortly after, translation. However, saccharides can also react with proteins non-enzymatically, a process termed glycation, which can cause impaired function and improper folding. Glycated proteins further react to form advanced glycation end-products, which have been implicated in the pathogenesis and progress of many diseases.Due to this pathological effect, glycation has been studied as a potential biomarker of these diseases. Photoaffinity labelling is a technique that is used to investigate the structure, and presence, of biological molecules; a precedent exists for its use in the study of carbohydrates in biological systems.Chapter 1 outlines the background of this thesis exploring previous studies of glycation, its effects, and methods used in recognition and photoaffinity labelling.Chapter 2 details the design and synthesis of a novel photoaffinity probe, and the optimisation of this synthesis. The target molecule was successfully produced and simpler alternatives to the initial synthetic route with similar yields are discussed.In Chapter 3 the use of the photoaffinity probe is studied. Labelling trials were performed on three proteins: human serum albumin (HSA), macrophage migration inhibitory factor (MIF), and casein. Mass spectrometry showed that the experiments with both HSA and MIF were successful, while the procedure appeared to lead to degradation of casein. Additionally, our work into developing techniques for identifying labelled samples is detailed. A diol-doped electrophoresis gel was not successful created, however, staining protein samples in polyacrylamide gel electrophoresis with curcumin showed promise.Chapter 4 explores the electrochemistry of the photoaffinity probe and details the use of the probe in functionalising a fluorine doped tin oxide (FTO) glass electrode. Cyclic voltammograms of Alizarin Red S (ARS), obtained using a treated electrode, suggest that surface functionalisation was successful.
|Date of Award
|24 Jan 2018
|Tony James (Supervisor) & Steven Bull (Supervisor)
- Boronic acid sensor
- photoaffinity labelling