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Abstract

Background: Sickle Cell Anaemia (SCA) is a genetically-inherited class of haemoglobinopathies arising due to a point mutation in the nucleotides coding for the sixth amino-acid of the β-haemoglobin. During sickle cell haemoglobin (HbS) synthesis the amino acid "valine" gets incorporated in the growing protein chain at the site of "glutamic acid". At the final stages of protein folding to form the quaternary structure of HbS, the overall hydrophilic character of haemoglobin is affected. This instability causes obstruction in protein-protein interactions causing the HbS molecules to polymerise into long chains within the Red Blood cells (RBC). The formation of these long chain polymers of mutated HbS causes the distorted conformation of RBC turning it into sickled shape structures. The sickling of erythrocytes is the main cause of microcirculatory obstruction leading to painful vaso-occlusive crisis. Rationale: This study aims to target the improper folding of the HbS protein causing the overall imbalance in the otherwise neutral character of haemoglobin. This can be executed through partially unfolding the protein to expose the mutated base to the hydrophobic core. This will allow the residue to take an appropriate orientation preventing the polymerization of HbS and subsequent distortion of erythrocytes. Methods: Further to a preliminary study on HbA molecules, partial unfolding and refolding experiments were performed to identify and ensure that the quaternary structure of haemoglobin could be retained. The appropriate conformations of the modified HbS molecules were monitored through Circular Dichroism (CD) Spectrophotometry. Protein unfolding was carried out in the presence of Dimethyl Sulfoxide (DMSO); a mild denaturant. Unfolding was suspended at variable intervals of time by the addition of chloroform. Solubility tests were performed to look for aggregation due to polymerization. Results: It was previously identified that a change in structural conformation of HbS molecules was brought about by allowing unfolding for 36 to 48 hours. However the newly adopted conformation was not found to be analogous to that of the wild-type haemoglobin (HbA). The molecular stability was found to mimic wild-type characteristics. It was identified that the modified protein took relatively the same time through the C18 chromatographic column as compared to chromatographic tests performed on HbA molecules. Ideally HbA molecules are completely soluble in buffer (pH 7). The modified proteins were found to be insoluble in potassium phosphate buffer at room temperature, however there was no formation of aggregates which indicates that no polymerisation had taken place. Conclusion: Through this study it is evident that controlled unfolding of HbS to expose the mutated residue to the hydrophobic core of the protein is able to restore lost stability. However, the appropriate refolding of the protein is also important in order to restore the functionality of haemoglobin as its quaternary structure is a requisite for oxygen binding and transport. Thus, although unfolding of the protein may be successful in giving HbS a stable configuration, more refined protocols for arresting unfolding or supporting appropriate refolding is essential in order to ensure that the structural conformation of the protein is retained.

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/content/papers/10.5339/qfarc.2014.HBPP0572
2014-11-18
2020-06-06
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http://instance.metastore.ingenta.com/content/papers/10.5339/qfarc.2014.HBPP0572
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