Effects of sucrose on rFVIIa aggregation and methionine oxidation
Effects of sucrose on rFVIIa aggregation and methionine oxidation
复制标题
DOI：
10.1016/j.ejps.2003.12.010
复制
发表时间：
2004-04-01
影响因子：
4.6
通讯作者：
Frokjaer, S
中科院分区：
文献类型：
--
作者：
Soenderkaer, S;Carpenter, JF;Frokjaer, S
本研究的目的是研究蔗糖对重组因子VIIa(RFVIIa)稳定性的影响，特别是聚集和蛋氨酸氧化的影响，以及不同环境条件对rFVIIa构象的影响。研究了rFVIIa在pH为5时的稳定性。用尺寸排除法测定了rFVIIa的聚集性，用反相高效液相色谱法测定了蛋氨酸氧化产物的生成。用傅里叶变换红外光谱(FTIR)和圆二色谱(CD)研究了蛋白质的构象。稳定性研究表明，增加蔗糖浓度减少了单体rFVIIa的损失，减少了二聚体/寡聚体和聚合物rFVIIa的形成。优先从蛋白质表面排除糖，将蛋白质分子种群从扩展的聚集能力物种转移到紧凑的自然状态，被认为是这些观察到的原因。RFVIIa对蛋氨酸氧化反应敏感，孵育形成两种单氧化和一种双氧化产物。与聚集不同，在蔗糖存在的情况下，蛋氨酸氧化被发现增加。两个容易氧化的蛋氨酸残基可能位于蛋白质表面。蔗糖存在时化学势的增加可能是氧化速率增加的原因。虽然FTIR光谱表明蔗糖引起rFVIIa结构的微小构象变化，但CD光谱不支持这一发现。与天然溶液的二级结构相比，沉淀rFVIIa的二级结构发生了变化。分子间β-折叠结构特征条带的出现与α-螺旋和分子内β-折叠结构的减少相一致。(C)2004爱思唯尔B.V.保留所有权利。
The aim of this study was to characterize the effects of sucrose on the stability of recombinant factor VIIa (rFVIIa), with special emphasis on aggregation and methionine oxidation, as well as to investigate the impact of various environmental conditions on the rFVIIa conformation. The stability of rFVIIa was studied at pH 5. Aggregation was monitored using size exclusion high-performance liquid chromatography (SE-HPLC), whereas formation of methionine oxidation products was measured by reversed-phase high-performance liquid chromatography (RP-HPLC). Fourier transform infrared (FTIR) spectroscopy and circular dichroism (CD) spectroscopy were used to study protein conformation. Stability studies showed that increasing sucrose concentrations reduced the loss of monomeric rFVIIa, and decreased formation of dimeric/oligomeric and polymeric rFVIIa. Preferential exclusion of the sugar from the protein's surface, which shifts the protein molecular population away from expanded aggregation competent species and toward the compact native state, is thought to account for these observations. rFVIIa is sensitive to methionine oxidation; two mono-oxidized and one di-oxidized product were formed upon incubation. Unlike aggregation, methionine oxidation was found to increase in the presence of sucrose. The two methionine residues susceptible to oxidation are presumably located at the protein surface. and the chemical potential increase in the presence of sucrose may account for the increase in oxidation rate. While FTIR spectroscopy suggested that sucrose induces small conformational changes in the rFVIIa structure, CD spectroscopy did not support this finding. The secondary structure of precipitated rFVIIa was changed when compared to the native solution secondary structure. Appearance of bands characteristic of intermolecular beta-sheet structure were found coincident with a decrease in alpha-helix and intramolecular beta-sheet structure. (C) 2004 Elsevier B.V. All rights reserved.