Oriented Immobilization of Proteins on Hydroxyapatite Surface Using Bifunctional Bisphosphonates as Linkers

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• 作者：Jivan N. Yewle ; Yinan Wei ; David A. Puleo ; Sylvia Daunert ; Leonidas G. Bachas
• 刊名：Biomacromolecules
• 出版年：2012
• 出版时间：June 11, 2012
• 年：2012
• 卷：13
• 期：6
• 页码：1742-1749
• 全文大小：342K
• 年卷期：v.13,no.6(June 11, 2012)
• ISSN：1526-4602

文摘

Oriented immobilization of proteins is an important step in creating protein-based functional materials. In this study, a method was developed to orient proteins on hydroxyapatite (HA) surfaces, a widely used bone implant material, to improve protein bioactivity by employing enhanced green fluorescent protein (EGFP) and 尾-lactamase as model proteins. These proteins have a serine or threonine at their N-terminus that was oxidized with periodate to obtain a single aldehyde group at the same location, which can be used for the site-specific immobilization of the protein. The HA surface was modified with bifunctional hydrazine bisphosphonates (HBPs) of various length and lipophilicity. The number of functional groups on the HBP-modified HA surface, determined by a 2,4,6-trinitrobenzenesulfonic acid (TNBS) assay, was found to be 2.8 脳 10^鈥? mol/mg of HA and unaffected by the length of HBPs. The oxidized proteins were immobilized on the HBP-modified HA surface in an oriented manner through formation of a hydrazone bond. The relative protein immobilization amounts through various HBPs were determined by fluorescence and bicinchoninic acid (BCA) assay and showed no significant effect by length and lipophilicity of HBPs. The relative amount of HBP-immobilized EGFP was found to be 10鈥?5 fold that of adsorbed EGFP, whereas the relative amount of 尾-lactamase immobilized through HBPs (2, 3, 4, 6, and 7) was not significantly different than adsorbed 尾-lactamase. The enzymatic activity of HBP-immobilized 尾-lactamase was measured with cefazolin as substrate, and it was found that the catalytic efficiency of HBP-immobilized 尾-lactamase improved 2鈥? fold over adsorbed 尾-lactamase. The results obtained demonstrate the feasibility of our oriented immobilization approach and showed an increased activity of the oriented proteins in comparison with adsorbed proteins on the same hydroxyapatite surface matrix.