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    Please use this identifier to cite or link to this item: http://ir.lib.ncu.edu.tw/handle/987654321/64950


    Title: 利用小角度中子及X光散射研究聚乙烯二醇化人類副甲狀腺素荷爾蒙(1-34)於溶液之結構;Structural Study of MonoPEGylated Human Parathyroid Hormone Fragments hPTH(1-34) in Solution Revealed by Small-Angle Neutron and X-ray Scattering
    Authors: 柳芝螢;Liu,Chih-Ying
    Contributors: 化學工程與材料工程學系
    Keywords: 小角度散射;人類副甲狀腺素荷爾蒙;聚乙烯二醇化;SANS;Human Parathyroid Hormone;PEGylation
    Date: 2014-07-21
    Issue Date: 2014-10-15 14:36:14 (UTC+8)
    Publisher: 國立中央大學
    Abstract: 聚乙烯二醇化 (PEGylation) 的胜肽或蛋白質藥物之物性、化性與生物活性取決於其接枝的聚乙烯二醇 (poly(ethylene glycol), PEG) 以及胜肽或蛋白質本身的結構。因此,了解聚乙烯二醇化的胜肽或蛋白質藥物之結構以及聚乙烯二醇和藥物之間之交互作用是相當重要的課題。本研究利用小角度中子及X光散射 (SANS及SAXS)、原二色光譜儀 (CD)、原子力顯微鏡 (AFM) 及穿透式電子顯微鏡 (TEM),研究聚乙烯二醇化人類副甲狀腺素荷爾蒙(1-34) (human parathyroid hormone (1-34)) 於溶液之結構,探討聚乙烯二醇化對人類副甲狀腺素荷爾蒙(1-34)和聚乙烯二醇本身結構之影響,以及不同接枝位置對其結構之效應。小角度散射圖譜顯示,人類副甲狀腺素荷爾蒙(1-34)於濃度100 mM的磷酸鹽緩衝溶液 (pH7.4) 中產生聚集,且聚集物之大小隨著溫度上升而變小,此在溶液中變溫時對其結構影響之結果為AFM及TEM無法做到的。
    將人類副甲狀腺素荷爾蒙(1-34)接枝單一聚乙烯二醇 (PEG5K) 後,因聚乙烯二醇的親水性,會自組裝 (self-assembly) 形成核-殼結構 (core-shell structure),較親水的聚乙烯二醇位於殼層,減少較疏水的人類副甲狀腺素荷爾蒙(1-34)與溶液接觸之表面積。利用柱狀結構加上表面連接高斯鏈 (Gaussian chain) 之形狀因子對實驗曲線作擬合,可求得核-殼結構之詳細結構資訊。擬合結果顯示,PEG5K接枝於人類副甲狀腺素荷爾蒙(1-34) C端之所形成核-殼結構的聚集數 (aggregation number) 約為接枝於N端的3倍。此外,聚乙烯二醇與人類副甲狀腺素荷爾蒙(1-34)間之作用力,使聚乙烯二醇貼附在人類副甲狀腺素荷爾蒙(1-34)之表面,造成接上人類副甲狀腺素荷爾蒙(1-34)之聚乙烯二醇之旋轉半徑 (radius of gyration) 較未接枝之聚乙烯二醇小。透過熱力學計算,推斷此作用力可能來自於PEG5K與人類副甲狀腺素荷爾蒙(1-34)間之charge-dipole作用力。
    ;In this study, we apply small-angle neutron and X-ray scattering (SANS and SAXS), circular dichroism (CD), atomic force microscope (AFM) and transmission electron microscopy (TEM) to investigate the peptide structure and the conjugated PEG chain conformation of monoPEGylated human parathyroid hormone (1-34) (hPTH(1-34)) in phosphate buffer. The enthalpic and entropic contributions of the PEG chain and the peptide due to conjugation are estimated accordingly.
    The scattering results show that the hPTH(1-34) in 100 mM phosphate buffer (pH7.4) aggregate into clusters. After hPTH(1-34) conjugated with PEG chain, the conjugates self-assemble into a core-shell structure with the PEG chains in the corona surrounding the hPTH(1-34) core. The core-shell structure helps reducing the surface accessible solvent area of the peptide. With model fitting by the form factor of a cylindrical core with Gaussian chains attached to the surface, the detailed structural information of the core-shell structures formed by the monoPEGylated hPTH(1-34) is extracted. The fitting results show that the conjugated PEG chain forms a collapsed conformation. This deformation suggests that the conjugate PEG interacts favorably with the peptide, so that part of the PEG chain bound to the surface of the peptide core instead of forming an unperturbed PEG coil adjacent to it. Moreover, the size of core-shell structure formed by the C-terminally monoPEGylated hPTH(1-34) is about three times larger than that of the N-terminally monoPEGylated ones. The different aggregation numbers of the self-assembled structures, triggered by different PEGylation sites, are reported. These size discrepancy due to different PEGylation sites could potentially affect the pharmacokinetics of the hPTH(1-34) drug. Therefore, the detailed structural analyses are important for development of “biobetters”.
    Appears in Collections:[化學工程與材料工程研究所] 博碩士論文

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