Intermediate formation at lower urea concentration in 'B' isomer of human serum albumin: a case study using domain specific ligands.

The urea-induced unfolding of 'N' isomer (occurring at pH 7.0) and 'B' isomer (occurring at pH 9.0) of human serum albumin was studied by fluorescence and circular dichroism spectroscopic measurements. Urea-induced destabilization in different domains of both the isomers was monitored by using domain specific ligands, hemin (domain-I), chloroform, bilirubin (domain-II), and diazepam (domain-III). Urea-induced denaturation of N and B isomers of HSA showed a two-step, three-state transition with accumulation of intermediates around 4.8-5.2M and 3.0-3.4M urea concentrations, respectively. During first transition (0-4.8M urea for N isomer and 0-3.0M urea for B isomer) a continuous decrease in diazepam binding suggested major conformational changes in domain-III prior to intermediate formation. On the other hand, binding of hemin, a ligand for domain-IB and chloroform, whose binding site is located in domain-IIA remains unchanged up to 5.0M urea for N isomer and 3.0M urea for B isomer. Similarly, fluorescence intensity of Trp-214 that resides in domain-IIA remained unchanged up to the above-said urea concentrations and decreased thereafter. Absence of any decrease in hemin binding, chloroform binding, and Trp-214 fluorescence suggested the non-involvement of domain-IB and domain-IIA in intermediate formation. A significant increase in bilirubin binding prior to intermediate formation showed favorable conformational rearrangement in bilirubin binding cavity formed by loop 4 of domain-IB and loop 3 of domain-IIA. Further, a nearly complete abolishment of bilirubin binding to both isomers around 7.0M and 6.0M urea concentrations, respectively, indicated complete separation of domain-I from domain-II from each other. From these observations it can be concluded that N to B transition of human serum albumin shifted the intermediate formation towards lower urea concentration (3.0-3.4M urea for B isomer as against 4.8-5.2M urea for N isomer). Further both the intermediates were found to possess similar alpha-helical (approximately 39%) content and ligand binding properties.