Intercalation of proteins into α-Zirconium phosphonates: tuning the binding affinities with phosphonate functions

Novel bioactive materials are prepared by the intercalation of proteins in the galleries of α-Zirconium phosphonates. α-Zirconium carboxymethylphosphonate (α-Zr(O3PCH2COOH)2 · xH2O), (α-ZrCMP), and α-Zirconium carboxyethylphosphonate (α-Zr(O3PCH2CH2COOH)2 · xH2O), (α-ZrCEP) indicated distinct affinities for four different proteins. A general procedure is described for the intercalation of Lysozyme (Lyz), myoglobin (Mb), hemoglobin (Hb), and glucose oxidase (GO) at the galleries of the above phosphonates, in aqueous solutions, at ambient temperature and at neutral pH. Binding stoichiometries were in the range of 1:35 (Lyz to α-ZrCMP) to 1:16 600 (Hb to α-ZrCEP) and these are more sparse when compared to the stoichiometries reported for intercalation in α-Zr(HPO4) · H2O (abbreviated as α-ZrP). Equilibrium binding constants (Kb) of these proteins depended on the protein as well as the Zr(IV) phosphonate type but varied from 3 × 104 (GO/α-ZrCEP) to 5.4 × 106 M−1 (Hb/α-ZrP). Powder X-ray diffraction (XRD) patterns of protein/α-ZrRP materials exhibited large interlayer spacings (38 Å for Lyz/α-ZrCMP to 54 Å for GO/α-ZrCEP). These enhanced d-spacings strongly support protein intercalation in the galleries. Circular dichroism (CD) and attenuated total reflectance FTIR (ATR-FTIR) methods have been used to investigate changes in the conformation of the proteins bound in the galleries. These spectral data indicated that the protein native conformations have been retained to a large extent. The active sites of the bound proteins are assessed in enzyme activity assays. Peroxidase-like activities of Mb and Hb decreased upon intercalation in the phosphonate galleries when compared to the activities of the corresponding proteins in α-ZrP. GO/α-ZrCEP, in contrast, exhibited enhanced activities when compared to the free enzyme. Subtle changes in the trends of binding affinities, the extent of structure retention, and enhanced activities suggest the strong role of interactions between the solid and the proteins in controlling the behavior of the intercalated proteins. The carboxylate functions of the α-ZrRP matrix, however, reduced the binding affinities of the four proteins studied here. The strong differences observed between proteins bound to α-ZrCMP and α-ZrCEP indicate the subtle role of the orientation of the carbonyl groups of these solids in their interactions with the bound proteins.