Guanidine hydrochloride denaturation of human serum albumin originates by local unfolding of some stable loops in domain III.
The effect of guanidine hydrochloride (GnHCl) on the global stability of human serum albumin (HSA) has been studied by fluorescence and circular dichroism spectroscopic measurements. The differential stability of native conformation of three HSA domains were explored by using domain-specific ligands, hemin (domain I), chloroform (domain II), bilirubin (at domain I/domain II interface) and diazepam (domain III). GnHCl induced unfolding transition curves as monitored by probes for secondary and tertiary structures were cooperative but noncoincidental. A strong ANS binding to the protein was observed around 1.8 M GnHCl, suggesting existence of intermediate states in the unfolding pathway of HSA. A gradual decrease (in the GnHCl concentration range 0.0-1.8 M) in the binding of diazepam indicates that domain III is the most labile to GnHCl denaturation. A significant increase in the binding of bilirubin up to 1.4 M GnHCl and decrease thereafter leading to complete abolishment of bilirubin binding at around 2.0 M GnHCl suggest favorable rearrangement and separation of domains I and II at 1.4 and 2.0 M GnHCl concentration, respectively. Above 1.6 M GnHCl, decrease of the binding of hemin, a ligand for domain I, chloroform, which binds in domain II and lone tryptophanyl fluorescence (Trp-214 located in domain II) indicate that at higher concentration of GnHCl domains I and II start unfolding simultaneously but the stability of domain I (7.4 Kcal/mol) is much more than domain II (4.3 Kcal/mol). A pictorial model for the unfolding of HSA domains, consistent with all these results, has been formulated, suggesting that domain III is the most labile followed by domain II while domain I is the most stable. A molten globule like state of domain III around 1.8 M GnHCl has also been identified and characterized.
Ahmad
Basir
B
Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh-202002, India.
Ahmed
Md Zulfazal
MZ
Haq
Soghra Khatun
SK
Khan
Rizwan Hasan
RH
D001663:Bilirubin / Q000378:metabolism; D001665:Binding Sites; D002725:Chloroform / Q000378:metabolism; D002942:Circular Dichroism; D003975:Diazepam / Q000378:metabolism; D019791:Guanidine / Q000737:chemistry*; D006427:Hemin / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012709:Serum Albumin / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D012709:Serum Albumin; D006427:Hemin; D002725:Chloroform; D019791:Guanidine; D003975:Diazepam; D001663:Bilirubin