Quantifying Vitamin K-dependent Holoprotein Compaction caused by differential γ-carboxylation using HPSEC

Authors

• Nicholas C. Vanderslice, University of Nebraska-LincolnFollow
• Amanda S. Messer, University of Nebraska-LincolnFollow
• Kanagasabai Vadivel, University of California, Los AngelesFollow
• S. Paul Bajaj, University of California, Los AngelesFollow
• Martin Phillips, University of California, Los AngelesFollow
• Mostafa Fatemi, University of Nebraska-LincolnFollow
• Weijie Xu, University of Nebraska-LincolnFollow
• William H. Velander, University of Nebraska-LincolnFollow

Date of this Version

2015

Document Type

Article

Citation

Anal Biochem. 2015 June 15; 479: 6–14.

Comments

© 2015 Published by Elsevier Inc.

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 -carboxyglutamic acid residues (Gla) are present in plasma-derived (pd-) and recombinant (r-) Factor IX (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 resulting in a 5-6% decrease in radius of hydration as observed by HPSEC. The filling of Ca²⁺ sites resulted greater compaction than for Mg²⁺ alone where this effect was additive or greater when both ions were present at physiologic levels. Less X²⁺ induced compaction was observed in r-FIX with lower Gla content populations which enabled the separation of biologically active from inactive r-FIX species by HPSEC. HPSEC was sensitive to R changes of ~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 X²⁺ avidity sites of the EGF1-like domain.