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

Abstract

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 β-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 × 10(-5) 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 10-15 fold that of adsorbed EGFP, whereas the relative amount of β-lactamase immobilized through HBPs (2, 3, 4, 6, and 7) was not significantly different than adsorbed β-lactamase. The enzymatic activity of HBP-immobilized β-lactamase was measured with cefazolin as substrate, and it was found that the catalytic efficiency of HBP-immobilized β-lactamase improved 2-5 fold over adsorbed β-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.

Recent advances in the synthesis, functionalization and biomedical applications of hydroxyapatite: a review

“…From the analysis of the results, they concluded that the oriented proteins showed better activity as compared to the absorbed proteins. 94 Furthermore, Li et al fabricated Gddoped luminescent HA and mesoporous SrHA nanorods and reported that their HA-modied aptamers could be used as a therapeutic agent against cancerous cells ( Fig. 9(c)).…”

Bisphosphonate-modified biomaterials for drug delivery and bone tissue engineering

“…An interesting extension of bonetargeting strategy involves a preparation of BP--X type of linkers, where X is a group or a ligand that can physically bind with high selectively to another group/ligand Y. Y can be a part of complex macromolecules, genetically engineered proteins or even cellular receptors ( Figure 1B). To date, this approach was utilized to enhance bone affinity of growth factors [52], to provide oriented immobilization of proteins with N-terminal serine residue [53] or avidin [54] to HAP surfaces, or even direct mesenchymal stem cells (MSCs) to bone [55]. Heparin [52], biotin [54] and integrin-binding peptidomimetic ligand [55] were used as ligand X to establish high-affinity physical binding to basic fibroblast growth factor, avidin and integrin a4b1 on the MSCs surface, respectively.…”

“…Heparin [52], biotin [54] and integrin-binding peptidomimetic ligand [55] were used as ligand X to establish high-affinity physical binding to basic fibroblast growth factor, avidin and integrin a4b1 on the MSCs surface, respectively. Moreover, a specific hydrazone linkage was formed between hydrazide group (as X group) of BP--X linker and aldehyde group generated on N-terminal of some suitable proteins to provide their oriented grafting [53]. The advantage of hydrazone-linked b-lactamase over the enzyme that was physically adsorbed to HAP was demonstrated by improved two to fivefold catalytic activity.…”

Immobilization of Enzymes on 2‐Hydroxyethyl Methacrylate and Glycidyl Methacrylate Copolymer Brushes

“…Poly(ethylene glycol) (PEG), [8] Pluronic, [9] bovine serum albumin (BSA), [10] dextran, [10] and other synthetic or natural polymers [11] have been used as spacers and have been proven to largely retain enzyme activity. The first is to introduce spacers between the materials and the proteins.…”

“…The spacers can allow the active sites on the substrate to be freely available, reduce unfavorable electrostatic or hydrophobic/hydrophilic interactions between the proteins and the materials, and increase conformational mobility of the enzymes. Poly(ethylene glycol) (PEG), [8] Pluronic, [9] bovine serum albumin (BSA), [10] dextran, [10] and other synthetic or natural polymers [11] have been used as spacers and have been proven to largely retain enzyme activity.…”