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

Nicholas C. Vanderslice, Amanda S. Messer, Kanagasabai Vadivel, S. Paul Bajaj, Martin Phillips, Mostafa Fatemi, Weijie Xu, William H. Velander

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

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 0.01 nm 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.

Original languageEnglish (US)
Pages (from-to)6-14
Number of pages9
JournalAnalytical Biochemistry
Volume479
DOIs
StatePublished - Jun 15 2015

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Carboxylation
Size exclusion chromatography
Vitamin K
Gel Chromatography
Compaction
Pressure
Binding Sites
Ions
Plasmas
Ultracentrifugation
Epidermal Growth Factor
Hydration
Metal ions
Metals
Acids
Population
Proteins

Keywords

  • Factor IX
  • High-pressure size exclusion
  • Vitamin K-dependent protein
  • chromatography
  • γ-Carboxylation

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Quantifying vitamin K-dependent holoprotein compaction caused by differential γ-carboxylation using high-pressure size exclusion chromatography. / Vanderslice, Nicholas C.; Messer, Amanda S.; Vadivel, Kanagasabai; Bajaj, S. Paul; Phillips, Martin; Fatemi, Mostafa; Xu, Weijie; Velander, William H.

In: Analytical Biochemistry, Vol. 479, 15.06.2015, p. 6-14.

Research output: Contribution to journalArticle

Vanderslice, Nicholas C. ; Messer, Amanda S. ; Vadivel, Kanagasabai ; Bajaj, S. Paul ; Phillips, Martin ; Fatemi, Mostafa ; Xu, Weijie ; Velander, William H. / Quantifying vitamin K-dependent holoprotein compaction caused by differential γ-carboxylation using high-pressure size exclusion chromatography. In: Analytical Biochemistry. 2015 ; Vol. 479. pp. 6-14.
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