Urea-induced denaturation process on defatted human serum albumin and in the presence of palmitic acid.
We report a study on the unfolding behavior of the most abundant protein contained in plasma, human serum albumin. The unfolding mechanisms in denaturing conditions induced by urea are studied for the defatted form (HSA) and for the palmitic acid:albumin (HSAPalm) complex. We employed the singular value decomposition method to determine the minimum number of structural states present in the unfolding processes. Low-resolution three-dimensional structures are reconstructed from the one-dimensional small-angle X-ray scattering patterns and are correlated with the parameters obtained from static and dynamic light scattering experiments. The unfolding process is pointed out by both ab initio and rigid body fitting methods that highlight a stepwise evolution of the protein structure toward open conformations. The superimpositions of the 3D structures provided independently by the two methods show very good agreements. The hydrodynamic radii estimated for the protein best fitting conformations are in satisfactory agreement with the experimental ones. The results show that the HSA unfolding process is consistent with previous spectroscopic studies that suggest a multistep unfolding pathway. In particular, a scheme in which domains I and II are opened in sequence and the presence of two intermediates are evidenced is presented. The opening sequence is different from that found using guanidine hydrochloride as denaturant agent. The stabilizing role of the fatty acids in the urea denaturation process is evident. The palmitic acid ligand strongly stabilizes the protein, which remains in the native form up to high denaturant concentrations. In this case, the unfolding process is characterized by a single-step mechanism.
Leggio
Claudia
C
Dipartimento di Chimica, Sapienza Universita di Roma, Piazzale A. Moro 5, I-00185 Roma, Italy.
Galantini
Luciano
L
Konarev
Peter V
PV
Pavel
Nicolae V
NV
D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008027:Light; D008958:Models, Molecular; D019308:Palmitic Acid / Q000737:chemistry*; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects; D015203:Reproducibility of Results; D012542:Scattering, Radiation; D053838:Scattering, Small Angle; D012709:Serum Albumin / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D014508:Urea / Q000494:pharmacology*; D014961:X-Ray Diffraction
D012709:Serum Albumin; D019308:Palmitic Acid; D014508:Urea