oa In-vitro assembly of a sickle cell haemoglobin intermediate to mimic wild-type characteristics

Sickle Cell Anaemia is an, autosomal, genetically-inherited, blood disorder, arising due to a point mutation in the bases coding for the sixth amino-acid of the β-chain of haemoglobin. The effects of the mutation begin to play role at the event of translation; during which the mutated haemoglobin (HbS) is synthesised. Here a hydrophobic protein residue (valine) is incorporated at the position of a hydrophilic residue (glutamic acid) in the growing protein chain. However, its orientation imitates that of the hydrophilic residue as otherwise seen in the wild type haemoglobin (HbA). Due to this change the hydrophobic side-chain of the amino-acid (valine) is prevented from being buried within the hydrophobic core of the protein and remains exposed to the surface. The resulting HbS thus displays a hydrophobic and unstable character with a tendency to polymerise with other HbS molecules causing the Red Blood Cells (RBC) to take a characteristic sickle shape. Through this research initiative, an attempt was made to unfold the protein to an extent that was sufficient to expose its hydrophobic core, thereby allowing it to engulf the side-chain through the formation of hydrophobic interactions. Thus the resultant modified protein would display an overall stable character, mimicking the wild-type HbA in spite of its mutational status. As a preliminary analysis, partial unfolding and refolding experiments were carried out on HbA molecules to determine whether the quaternary structure of haemoglobin would be retained. These experiments were monitored through Circular Dichroism (CD) Spectrophotometry. Partial unfolding was targeted by treating the protein with different concentrations of a mild denaturant (dimethyl sulfoxide). Unfolding was arrested at different stages of time (12, 24, 36, 48, 60, 72 hours) by the introduction of an organic solvent (chloroform) that induces precipitation of the protein. The modified protein was then tested for changes in hydrophobic character and stability by Reverse Phase Chromatography using C18 columns. Tests for solubility and aggregation were also performed spectrophotometrically. All tests were carried out under both oxygenated and deoxygenated conditions. It was observed that modified haemoglobin molecules arrested at 36 and 48 hours displayed a change in structural conformation. However, CD spectrophotometric analysis confirmed that they did not refold to resemble the wild-type HbA . Chromatographic results showed that the modified protein developed an overall neutral character, while spectrophotometric analysis proved that the molecules were insoluble in potassium phosphate buffer (pH 7) under both oxygenated and deoxygenated conditions. The study proved that partial unfolding up to 36 to 48 hours was sufficient to expose the hydrophobic core to trigger interactions that causes the haemoglobin molecule to attain an overall neutral and stable character. However, further analysis is ongoing to control the refolding of the protein to more closely match its native state. The study is also experimenting with other models of denaturants as well as more refined methods to arrest unfolding in order to improve solubility of the protein.