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 language | English |
|---|---|
| Pages (from-to) | 209-217 |
| Number of pages | 9 |
| Journal | Journal of Biological Inorganic Chemistry |
| Volume | 14 |
| Issue number | 2 |
| DOIs | |
| Publication status | Published - Feb 2009 |
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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 journal › Article
}
TY - JOUR
T1 - Reversible two-step unfolding of heme-human serum albumin
T2 - A 1H-NMR relaxometric and circular dichroism study
AU - Fanali, Gabriella
AU - De Sanctis, Giampiero
AU - Gioia, Magda
AU - Coletta, Massimo
AU - Ascenzi, Paolo
AU - Fasano, Mauro
PY - 2009/2
Y1 - 2009/2
N2 - 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.
AB - 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.
KW - H-NMR relaxometry
KW - Circular dichroism
KW - Folding intermediate state
KW - Guanidinium chloride
KW - Heme-human serum albumin
UR - http://www.scopus.com/inward/record.url?scp=58849167237&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=58849167237&partnerID=8YFLogxK
U2 - 10.1007/s00775-008-0439-7
DO - 10.1007/s00775-008-0439-7
M3 - Article
C2 - 18936983
AN - SCOPUS:58849167237
VL - 14
SP - 209
EP - 217
JO - Journal of Biological Inorganic Chemistry
JF - Journal of Biological Inorganic Chemistry
SN - 0949-8257
IS - 2
ER -