Iqbal, Z.; (2008) Electrochemical modulation of sickle cell haemoglobin polymerisation. Doctoral thesis , University of London.

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Abstract

Sickle cell haemoglobin differs from normal haemoglobin by a single amino acid in its chain. This amino acid replacement, from glutamic acid to valine, causes polymerisation of proteins into defined long insoluble fibres with a typical diameter of 21.5 nm. The polymerisation is triggered by the formation of deox haemoglobin from oxyhaemoglobin in low oxygen partial pressures, which results in a conformational change in the secondary structure of the protein. Pathogenesis in sickle cell disease depends on the polymerisation and gelation of deoxygenated HbS molecules. In this work, an electrochemical method has been described to modulate the oxygen concentration in an optically transparent thin layer cell to produce deoxyhaemoglobin whilst monitoring the extent of polymerisation using turbidity measurements. The oxygen was depleted in the vicinity of the electrode and triggered the polymerisation. The dependence of protein concentration, temperature, pH and ionic strength on the nucleation and elongation of HbS polymerisation was characterised at the electrode surface and the kinetics of polymerisation was investigated using a model for fibrillogenesis describing a two-step process of nucleation followed by elongation. The rate constants, determined for a number of conditions, showed that nucleation is far slower than the growth whilst polymerisation at the surface was demonstrated to occur in three stages with an initial time delay when no structures were observed followed b growth of fibrous hair-like strands and finally gel-like aggregation. An understanding of the factors which affect polymerisation at a surface and an insight into the dynamics and mechanism of polymer aggregation and the pathophysiology of sickle cell disease has been provided. A screening method for substances that effect the fibre nucleation and/or growth that could be valuable to the pharmaceutical industry for treating sickle cell disease is also presented.

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