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

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 -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 Ca 2+ and Mg 2+ binding sites resulting in a 5-6% decrease in radius of hydration as observed by HPSEC. The filling of Ca 2+ sites resulted greater compaction than for Mg 2+ alone where this effect was additive or greater when both ions were present at physiologic levels. Less X 2+ 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 X 2+ sites of the Gla domain and higher X 2+ avidity sites of the EGF1-like domain.