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 journalArticle

Abstract

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
Volume14
Issue number2
DOIs
Publication statusPublished - Feb 2009

Fingerprint

Circular Dichroism
Heme
Serum Albumin
Nuclear magnetic resonance
Myristic Acid
Guanidine
Conformations
Circular dichroism spectroscopy
Proton Magnetic Resonance Spectroscopy
Protein Unfolding
Denaturation
Scavenging
Absorption spectroscopy
Albumins
Spectrum Analysis
Fatty Acids
Binding Sites

Keywords

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

ASJC Scopus subject areas

  • Biochemistry
  • Inorganic Chemistry

Cite this

Reversible two-step unfolding of heme-human serum albumin : A 1H-NMR relaxometric and circular dichroism study. / Fanali, Gabriella; De Sanctis, Giampiero; Gioia, Magda; Coletta, Massimo; Ascenzi, Paolo; Fasano, Mauro.

In: Journal of Biological Inorganic Chemistry, Vol. 14, No. 2, 02.2009, p. 209-217.

Research output: Contribution to journalArticle

Fanali, Gabriella ; De Sanctis, Giampiero ; Gioia, Magda ; Coletta, Massimo ; Ascenzi, Paolo ; Fasano, Mauro. / Reversible two-step unfolding of heme-human serum albumin : A 1H-NMR relaxometric and circular dichroism study. In: Journal of Biological Inorganic Chemistry. 2009 ; Vol. 14, No. 2. pp. 209-217.
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