Investigation into pH – Dependent Conformational Isomerisation of Human Serum Albumin using Spectroscopic Techniques

Abstract

Human serum albumin (HSA) is a 585-residue, model all α-helical protein comprising three homologous domains (I, II, and III), which is present in the circulatory system and acts as a carrier for various kinds of ligands, metabolites, drugs, etc. Earlier literature reports have indicated that HSA undergoes reversible conformational transitions depending on the solution conditions such as changes in pH, temperature, and also in the presence of denaturants whereby non-covalent interactions such as hydrogen bonding, hydrophobic and van der Waals are disrupted but the disulphides remain intact. In this thesis, we have carried out a systematic study on the conformational isomerisation of HSA as a function of pH using fluorescence and CD spectroscopic techniques. The fluorescence intensity and anisotropy measurements were performed using intrinsic (single tryptophan, Trp-214) and extrinsic (1,8-anilinonaphthalene sulfonate, ANS, hydrophobicity reporter) fluorophores. Results obtained from our spectroscopic measurements suggested that HSA forms a partially-expanded, molten globule-like state at pH 3.5 with a concurrent loss of α-helical content during N↔F transition that appears to be a two-state transition. Additionally, our equilibrium unfolding studies utilizing both thermally- and chemically-induced denaturation of HSA revealed that the unfolding of the molten globule-like state, populated at pH 3.5, occurs in a non-cooperative manner and is less stable than the native HSA at neutral pH.