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

Gabriella Fanali, Giampiero De Sanctis, Magda Gioia, Massimo Coletta, Paolo Ascenzi, Mauro Fasano

Research output: Contribution to journalArticlepeer-review


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) × 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) × 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.

Original languageEnglish
Pages (from-to)209-217
Number of pages9
JournalJournal of Biological Inorganic Chemistry
Issue number2
Publication statusPublished - Feb 2009


  • H-NMR relaxometry
  • Circular dichroism
  • Folding intermediate state
  • Guanidinium chloride
  • Heme-human serum albumin

ASJC Scopus subject areas

  • Biochemistry
  • Inorganic Chemistry


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