This thesis is concerned with the loading of transferrin with manganese and some of its chemical and biological properties. Manganese is bound to transferrin as Mn(III) with a characteristic ligand (Tyr) to metal (Mn(III) charge-transfer band at a wavelength of 430 nm. Caeruloplasmin is shown to enhance the uptake of manganese from MnC1<sub>2</sub> by apo-hTf. However binding is often incomplete and slow. A novel method of loading hTf with Mn using KMnO<sub>4</sub> is reported. This method leads to rapid uptake and inductively coupled plasma atomic emission spectroscopy (ICP-AES) determinants confirmed the binding of at least two Mn per hTf molecule. The possible oxidising effects of MnO<sub>4</sub><sup>-</sup> on protein amino acid side chains was considered. In model systems MnO<sub>4</sub><sup>-</sup> oxidises methionine to methionine sulfoxide and methionine sulfone. Evidence of structural changes in apo-hTf induced by Mn(III) binding was obtained by studies using [e-<sup>13</sup>C]Met-hTf. Preliminary work suggests that Mn(III), like several other metals studied, preferentially binds to the C-lobe first, although this may result in an open domain conformation. Fe(III) as Fe(NTA)<sub>2</sub> was found to displace Mn(III) from hhTf but displacement was slower when hTf had been loaded using KMnO<sub>4</sub> rather than MnCl<sub>2</sub>. KMnO<sub>4</sub> was not able to displace Fe(III) from Fe<sub>2</sub>-hTf. Attempts to crystallise Mn-hTf to characterise these structural changes proved difficult. Crystals grew but were of poor quality and did not diffract. Many large crystals were obtained from solutions of Fe<sub>2</sub>-hTf. The crystals were red/orange and ellipsoidal in shape. Of the Fe<sub>2</sub>-hTf crystals grown, one diffracted to 3.3 Å with the data being complete to 90%, but not enough information was gained for adequate molecular replacement and structural solution.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:642218 |
| Date | January 2003 |
| Creators | Bunyan, Kerry Emma |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/15569 |
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