Quantifying vitamin K-dependent holoprotein compaction caused by differential γ-carboxylation using high-pressure size exclusion chromatography

This study uses high-pressure size exclusion chromatography (HPSEC) to quantify divalent metal ion (X2+)-induced compaction found in vitamin K-dependent (VKD) proteins. Multiple X2+ binding sites formed by the presence of up to 12 γ-carboxyglutamic acid (Gla) residues are present in plasma-derived F...

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Bibliographic Details
Published in:Analytical biochemistry 2015-06, Vol.479, p.6-14
Main Authors: Vanderslice, Nicholas C., Messer, Amanda S., Vadivel, Kanagasabai, Bajaj, S. Paul, Phillips, Martin, Fatemi, Mostafa, Xu, Weijie, Velander, William H.
Format: Article
Language:English
Subjects:
1-Carboxyglutamic Acid - chemistry
Binding Sites
Calcium - metabolism
Cations, Divalent - metabolism
Chromatography, Gel - methods
Factor IX
Factor IX - chemistry
Factor IX - metabolism
High-pressure size exclusion chromatography
Humans
Magnesium - metabolism
Models, Molecular
Protein Conformation
Recombinant Proteins - chemistry
Recombinant Proteins - metabolism
Vitamin K - metabolism
Vitamin K-dependent protein
γ-Carboxylation
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Summary:This study uses high-pressure size exclusion chromatography (HPSEC) to quantify divalent metal ion (X2+)-induced compaction found in vitamin K-dependent (VKD) proteins. Multiple X2+ binding sites formed by the presence of up to 12 γ-carboxyglutamic acid (Gla) residues are present in plasma-derived FIX (pd-FIX) and recombinant FIX (r-FIX). Analytical ultracentrifugation (AUC) was used to calibrate the Stokes radius (R) measured by HPSEC. A compaction of pd-FIX caused by the filling of Ca2+ and Mg2+ binding sites resulted in a 5 to 6% decrease in radius of hydration as observed by HPSEC. The filling of Ca2+ sites resulted in greater compaction than for Mg2+ alone where this effect was additive or greater when both ions were present at physiological levels. Less X2+-induced compaction was observed in r-FIX with lower Gla content populations, which enabled the separation of biologically active r-FIX species from inactive ones by HPSEC. HPSEC was sensitive to R changes of approximately 0.01nm that enabled the detection of FIX compaction that was likely cooperative in nature between lower avidity X2+ sites of the Gla domain and higher avidity X2+ sites of the epidermal growth factor 1 (EGF1)-like domain.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2015.03.019