Intermediate formation at lower urea concentration in 'B' isomer of human serum albumin: a case study using domain specific ligands
| Autor: | Rizwan Hasan Khan, Mohd Khursheed Alam Khan, Basir Ahmad, Soghra Khatun Haq |
|---|---|
| Rok vydání: | 2004 |
| Předmět: |
Circular dichroism
Protein Denaturation Protein Folding Stereochemistry Protein Conformation Biophysics Biochemistry chemistry.chemical_compound Structure-Activity Relationship Isomerism medicine Humans Protein Isoforms Urea Denaturation (biochemistry) Binding site Molecular Biology Serum Albumin Diazepam binding Binding Sites Diazepam Bilirubin Cell Biology Ligand (biochemistry) Human serum albumin Protein Structure Tertiary chemistry Hemin Chloroform medicine.drug Protein Binding |
| Zdroj: | Biochemical and biophysical research communications. 314(1) |
| ISSN: | 0006-291X |
| Popis: | 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 4.8–5.2 M and 3.0–3.4 M urea concentrations, respectively. During first transition (0–4.8 M urea for N isomer and 0–3.0 M 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 5.0 M urea for N isomer and 3.0 M 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 7.0 M and 6.0 M 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 (3.0–3.4 M urea for B isomer as against 4.8–5.2 M urea for N isomer). Further both the intermediates were found to possess similar α-helical (∼39%) content and ligand binding properties. |
| Databáze: | OpenAIRE |
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