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| Predicate | Object |
|---|---|
| rdf:type | |
| lifeskim:mentions | |
| pubmed:issue |
20
|
| pubmed:dateCreated |
1998-6-22
|
| pubmed:abstractText |
The binding of the multidomain protein factor VIIa (fVIIa) to tissue factor provides the interprotein communication necessary to make fVIIa an efficient catalyst of the initial event in the extrinsic pathway of blood coagulation. We have investigated the stability of individual domains in fVIIa and the influence of Ca2+ and an irreversible active-site inhibitor (FFR-chloromethyl ketone). Equilibrium guanidine hydrochloride (GuHCl)-induced unfolding monitored by tryptophan fluorescence and far-UV circular dichroism (CD) demonstrated that the gamma-carboxyglutamic acid (Gla) domain unfolds at 0.3 M GuHCl and the serine protease (SP) domain at 3 M GuHCl and that Ca2+ is a prerequisite for the formation of an ordered, compact structure in the Gla domain. The loss of amidolytic activity coincides with the first transition, which is stabilized by the active-site inhibitor, and a change in the environment of the active site is demonstrated using a fluorescent inhibitor (DEGR-chloromethyl ketone). Thermal unfolding monitored by differential scanning calorimetry (DSC) reveals that Ca2+ stabilizes the SP domain slightly, increasing the unfolding temperature by 2.7 degrees C. In addition, Ca2+ is required for a large enthalpy change concomitant with unfolding of the Gla domain, and this unfolding enthalpy is only detectable in the presence of the SP domain, indicating some kind of interaction between these domains. Thermal unfolding measured by CD indicates secondary structural changes at the same temperature as the heat absorption in the DSC but only when both the Gla domain and the SP domain are present together with Ca2+ ions. Taken together, these results indicate a Ca2+-dependent interaction between the Gla domain and the SP domain, implying a high degree of flexibility of the domains in free fVIIa. It is also shown that the epidermal growth factor-like domains are stable at elevated temperatures and high GuHCl concentrations. Moreover, already at physiological temperature, subtle structural changes take place which influence the overall shape of fVIIa and are detrimental to its enzymatic activity.
|
| pubmed:language |
eng
|
| pubmed:journal | |
| pubmed:citationSubset |
IM
|
| pubmed:chemical | |
| pubmed:status |
MEDLINE
|
| pubmed:month |
May
|
| pubmed:issn |
0006-2960
|
| pubmed:author | |
| pubmed:issnType |
Print
|
| pubmed:day |
19
|
| pubmed:volume |
37
|
| pubmed:owner |
NLM
|
| pubmed:authorsComplete |
Y
|
| pubmed:pagination |
7203-12
|
| pubmed:dateRevised |
2004-11-17
|
| pubmed:meshHeading |
pubmed-meshheading:9585532-Acrylamide,
pubmed-meshheading:9585532-Acrylamides,
pubmed-meshheading:9585532-Calorimetry, Differential Scanning,
pubmed-meshheading:9585532-Circular Dichroism,
pubmed-meshheading:9585532-Factor VIIa,
pubmed-meshheading:9585532-Guanidine,
pubmed-meshheading:9585532-Humans,
pubmed-meshheading:9585532-Light,
pubmed-meshheading:9585532-Models, Molecular,
pubmed-meshheading:9585532-Protein Conformation,
pubmed-meshheading:9585532-Protein Denaturation,
pubmed-meshheading:9585532-Protein Structure, Secondary,
pubmed-meshheading:9585532-Protein Structure, Tertiary,
pubmed-meshheading:9585532-Scattering, Radiation,
pubmed-meshheading:9585532-Spectrometry, Fluorescence,
pubmed-meshheading:9585532-Thermodynamics
|
| pubmed:year |
1998
|
| pubmed:articleTitle |
Conformational stability of factor VIIa: biophysical studies of thermal and guanidine hydrochloride-induced denaturation.
|
| pubmed:affiliation |
Vessel Wall Biology, Health Care Discovery, Novo Nordisk A/S, Gentofte, Denmark. pof@novo.dk
|
| pubmed:publicationType |
Journal Article
|