Quantifying vitamin K-dependent holoprotein compaction caused by differential γ-carboxylation using high-pressure size exclusion chromatography.
Quantifying vitamin K-dependent holoprotein compaction caused by differential γ-carboxylation using high-pressure size exclusion chromatography.
复制标题
使用高压尺寸排阻色谱法量化由差异γ-羧化引起的维生素 K 依赖性全蛋白压缩。
DOI：
10.1016/j.ab.2015.03.019
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发表时间：
2015
影响因子：
2.9
通讯作者：
Velander,WilliamH
中科院分区：
文献类型：
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作者：
Vanderslice,NicholasC;Messer,AmandaS;Vadivel,Kanagasabai;Bajaj,SPaul;Phillips,Martin;Fatemi,Mostafa;Xu,Weijie;Velander,WilliamH
本研究使用高压尺寸排阻色谱法（HPSEC）来定量二价金属离子（X2+）诱导的维生素K依赖性（VKD）蛋白质的压缩。血浆源性FIX（pd-FIX）和重组FIX（r-FIX）中存在多达12个γ-羧基谷氨酸（Gla）残基形成的多个X2+结合位点。使用分析超离心（AUC）来校准通过HPSEC测量的斯托克斯半径（R）。通过HPSEC观察到，由Ca 2+和Mg 2+结合位点填充引起的pd-FIX压实导致水合半径降低5 - 6%。填充的Ca 2+网站导致更大的压实比单独的Mg 2+，这种效果是加性的或更大的，当两种离子都存在于生理水平。在Gla含量较低的r-FIX群体中观察到较少的X2+诱导的压实，这使得能够通过HPSEC将具有生物活性的r-FIX物质与无活性物质分离。HPSEC对约0.01 nm的R变化敏感，这使得能够检测到FIX压实，其可能在Gla结构域的较低亲合力X2+位点和表皮生长因子1（EGF 1）样结构域的较高亲合力X2+位点之间具有协同作用。
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 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 Ca2+and Mg2+binding sites resulted in a 5 to 6% decrease in radius of hydration as observed by HPSEC. The filling of Ca2+sites resulted in greater compaction than for Mg2+alone where this effect was additive or greater when both ions were present at physiological levels. Less X2+-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 0.01 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 avidity X2+sites of the epidermal growth factor 1 (EGF1)-like domain.