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Metal-Enzyme Frameworks: Role of Metal Ions in Promoting Enzyme Self-Assembly on α‑Zirconium(IV) Phosphate Nanoplates

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journal contribution
posted on 05.03.2013 by Ajith Pattammattel, Inoka K. Deshapriya, Ruma Chowdhury, Challa V. Kumar
Previously, an ion-coupled protein binding (ICPB) model was proposed to explain the thermodynamics of protein binding to negatively charged α-Zr­(IV) phosphate (α-ZrP). This model is tested here using glucose oxidase (GO) and met-hemoglobin (Hb) and several cations (Zr­(IV), Cr­(III), Au­(III), Al­(III), Ca­(II), Mg­(II), Zn­(II), Ni­(II), Na­(I), and H­(I)). The binding constant of GO with α-ZrP was increased ∼380-fold by the addition of either 1 mM Zr­(IV) or 1 mM Ca­(II), and affinities followed the trend Zr­(IV) ≃ Ca­(II) > Cr­(III) > Mg­(II) ≫ H­(I) > Na­(I). Binding studies could not be conducted with Au­(III), Al­(III), Zn­(II), Cu­(II), and Ni­(II), as these precipitated both proteins. Zr­(IV) increased Hb binding constant to α-ZrP by 43-fold, and affinity enhancements followed the trend Zr­(IV) > H­(I) > Mg­(II) > Na­(I) > Ca­(II) > Cr­(III). Zeta potential studies clearly showed metal ion binding to α-ZrP and affinities followed the trend, Zr­(IV) ≫ Cr­(III) > Zn­(II) > Ni­(II) > Mg­(II) > Ca­(II) > Au­(III) > Na­(I) > H­(I). Electron microscopy showed highly ordered structures of protein/metal/α-ZrP intercalates on micrometer length scales, and protein intercalation was also confirmed by powder X-ray diffraction. Specific activities of GO/Zr­(IV)/α-ZrP and Hb/Zr­(IV)/α-ZrP ternary complexes were 2.0 × 10–3 and 6.5 × 10–4 M–1 s–1, respectively. While activities of all GO/cation/α-ZrP samples were comparable, those of Hb/cation/α-ZrP followed the trend Mg­(II) > Na­(I) > H­(I) > Cr­(III) > Ca­(II) ≃ Zr­(IV). Metal ions enhanced protein binding by orders of magnitude, as predicted by the ICPB model, and binding enhancements depended on charge as well as the phosphophilicity/oxophilicity of the cation.