Reversible two-step unfolding of heme-human serum albumin: a ¹H-NMR relaxometric and circular dichroism study

• Published in: Journal of biological inorganic chemistry Vol. 14; no. 2; pp. 209 - 217
• Main Authors: Fanali, Gabriella, De Sanctis, Giampiero, Gioia, Magda, Coletta, Massimo, Ascenzi, Paolo, Fasano, Mauro
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Berlin/Heidelberg : Springer-Verlag 2009; Springer-Verlag
• Subjects: Biochemistry; Biomedical and Life Sciences; Life Sciences; Microbiology; Original Paper; Folding intermediate state; Guanidinium chloride; H-NMR relaxometry; Heme–human serum albumin; Circular dichroism
• ISSN: 0949-8257; 1432-1327
• DOI: 10.1007/s00775-008-0439-7

Human serum albumin (HSA) participates in heme scavenging, the bound heme turning out to be a reactivity center and a powerful spectroscopic probe. Here, the reversible unfolding of heme-HSA has been investigated by ¹H-NMR relaxometry, circular dichroism, and absorption spectroscopy. In the presence of 6 equiv of myristate (thus fully saturating all available fatty acid binding sites in serum heme-albumin), 1.0 M guanidinium chloride induces some unfolding of heme-HSA, leading to the formation of a folding intermediate; this species is characterized by increased relaxivity and enhanced dichroism signal in the Soret region, suggesting a more compact heme pocket conformation. Heme binds to the folding intermediate with K d = (1.2 ± 0.1) x 10⁻⁶ M. In the absence of myristate, the conformation of the folding intermediate state is destabilized and heme binding is weakened [K d = (3.4 ± 0.1) x 10⁻⁵ M]. Further addition of guanidinium chloride (up to 5 M) brings about the usual denaturation process. In conclusion, myristate protects HSA from unfolding, stabilizing a folding intermediate state in equilibrium with the native and the fully unfolded protein, envisaging a two-step unfolding pathway for heme-HSA in the presence of myristate.