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 (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. © 2015 Published by Elsevier Inc. Corresponding Author. William H Velander, 207 Othmer Hall, 820 N. 16th St., University of Nebraska-Lincoln, Lincoln, NE 68588-0668; Tel: 402-472-3697, Fax: 402-472-6989; wvelander2@unl.edu. Nicholas Vanderslice, vanderslice.nicholas@gmail.com Amanda S. Messer, asmesser3@gmail.com Kanagasabai Vadivel, kvadivel@mednet.ucla.edu S. Paul Bajaj, pbajaj@mednet.ucla.edu Martin Phillips, mlphill@ucla.edu Mostafa Fatemi, mfatemi2@unl.edu Weijie Xu, weijiexuchn@gmail.com Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. HHS Public Access Author manuscript Anal Biochem. Author manuscript; available in PMC 2016 June 15. Published in final edited form as: Anal Biochem. 2015 June 15; 479: 6–14. doi:10.1016/j.ab.2015.03.019. A uhor M anscript