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 alpha-Zirconium phosphonates. alpha-Zirconium carboxvmethylphosphonate (alpha-Zr(O3PCH2COOH)(2)(.)xH(2)O), (alpha-ZrCMP), and alpha-Zirconium carboxyeihylphosphonate (alpha-Zr(O3PCH2CH2COOH)(2) (.) xH(2)O), (alpha-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 1:15 (Lyz to alpha-ZrCMP) to 1:16 600 (Hb to alpha-ZrCEP) and these are more sparse when compared to the stoichiometfies reported for inter-calation in alpha-Zr(HPO4) (.) H2O (abbreviated as alpha-ZrP). Equilibrium binding constants (K-b) of these proteins depended on the protein as well as the Zr(IV) phosphonate type but varied frorn 3 x 10(4) (GO/alpha-ZrCEP) to 5.4 x 10(6) M-1 (Hb/alpha-ZrP). Powder X-ray diffraction (XRD) patterns of protein/alpha-ZrRP materials exhibited large interlayer spacings (38Angstrom for Lyz/alpha-ZrCMP to 54Angstrom for GO/alpha-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 alpha-ZrP. GO/alpha-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 alpha-ZrRP matrix, however, reduced the binding affinities of the four proteins studied here. The strong differences observed between proteins bound to alpha-ZrMP and alpha-ZrCEP indicate the subtle role of the orientation of the carbonyl groups of these solids in their interactions with the bound proteins. (C) 2004 Elsevier Inc. All rights reserved.