title: Quantifying vitamin K-dependent holoprotein compaction caused by differential γ-carboxylation using high-pressure size exclusion chromatography.
abstract: This study uses high-pressure size exclusion chromatography (HPSEC) to quantify divalent metal ion (X(2+))-induced compaction found in vitamin K-dependent (VKD) proteins. Multiple X(2+) 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 Ca(2+) and Mg(2+) binding sites resulted in a 5 to 6% decrease in radius of hydration as observed by HPSEC. The filling of Ca(2+) sites resulted in greater compaction than for Mg(2+) alone where this effect was additive or greater when both ions were present at physiological levels. Less X(2+)-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 X(2+) sites of the Gla domain and higher avidity X(2+) sites of the epidermal growth factor 1 (EGF1)-like domain.
authors: Protein Purification and Characterization Laboratories, Department of Chemical and Biomolecular Engineering, University of Nebraska-Lincoln, Lincoln, NE 68588, USA.
authors: Nicholas C
authors: NC
authors: Vanderslice
authors: Protein Purification and Characterization Laboratories, Department of Chemical and Biomolecular Engineering, University of Nebraska-Lincoln, Lincoln, NE 68588, USA; Protein Science Laboratory, UCLA/Orthopaedic Hospital, Department of Orthopaedic Surgery and Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA.
authors: Amanda S
authors: AS
authors: Messer
authors: Protein Science Laboratory, UCLA/Orthopaedic Hospital, Department of Orthopaedic Surgery and Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA.
authors: Kanagasabai
authors: K
authors: Vadivel
authors: Protein Science Laboratory, UCLA/Orthopaedic Hospital, Department of Orthopaedic Surgery and Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA 90095, USA.
authors: S Paul
authors: SP
authors: Bajaj
authors: UCLA-DOE Biochemistry Instrumentation Facility, Department of Chemistry and Biochemistry, University of California, Los Angeles, Los Angeles, CA 90095, USA.
authors: Martin
authors: M
authors: Phillips
authors: Protein Purification and Characterization Laboratories, Department of Chemical and Biomolecular Engineering, University of Nebraska-Lincoln, Lincoln, NE 68588, USA.
authors: Mostafa
authors: M
authors: Fatemi
authors: Protein Purification and Characterization Laboratories, Department of Chemical and Biomolecular Engineering, University of Nebraska-Lincoln, Lincoln, NE 68588, USA.
authors: Weijie
authors: W
authors: Xu
authors: Protein Purification and Characterization Laboratories, Department of Chemical and Biomolecular Engineering, University of Nebraska-Lincoln, Lincoln, NE 68588, USA. Electronic address: wvelander2@unl.edu.
authors: William H
authors: WH
authors: Velander
mesh_terms: D015055:1-Carboxyglutamic Acid / Q000737:chemistry*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002413:Cations, Divalent / Q000378:metabolism; D002850:Chromatography, Gel / Q000379:methods*; D005164:Factor IX / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D008958:Models, Molecular; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014812:Vitamin K / Q000378:metabolism
chemical_list: D002413:Cations, Divalent; D011994:Recombinant Proteins; D014812:Vitamin K; D015055:1-Carboxyglutamic Acid; D005164:Factor IX; D008274:Magnesium; D002118:Calcium
keywords: Factor IX; High-pressure size exclusion chromatography; Vitamin K-dependent protein; γ-Carboxylation