Chemical and Biomolecular Engineering, Department of


Date of this Version


Document Type



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


© 2015 Published by Elsevier Inc.


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.