Metal-Enzyme Frameworks: Role of Metal Ions in Promoting Enzyme Self-Assembly on α‑Zirconium(IV) Phosphate Nanoplates Ajith Pattammattel, Inoka K. Deshapriya, Ruma Chowdhury, and Challa V. Kumar* Departments of Chemistry and Molecular & Cell Biology, University of Connecticut, U-3060, Storrs, Connecticut 06269, United States * S Supporting Information ABSTRACT: 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. 1. INTRODUCTION Protein self-assembly at liquid−solid interfaces is of current interest, and this is often achieved via chemical, 1 biomolecular, 2 thermal, 3 or metal-induced 4−6 assembly. Protein self-assembly is challenging because of the large size of proteins, multiple functional groups on their surfaces, their fragility to solvents, sensitivity to particular ions and extreme pH, and their vulnerability to degradation by proteases, which are ubiquitous. Protein assemblies are increasingly being used in biosensing, 7 biomaterials, 8 biocatalysis, 9 and biomedicine. 10 Therefore, it is critical to understand how such assemblies can be constructed by a systematic approach and establish the details of the mechanism of protein assembly, so that protein assembly can be controlled in a rational, predictable manner. Despite the widespread interest in the application of proteins bound to solid surfaces, there are no quantitative models or rational approaches to address these important issues. The mechanism of protein binding to solid surfaces is complex, not fully understood, 11 but in the case of most water- soluble, charged proteins, protein binding requires charge neutralization at the protein−solid interface, and this electro- static requirement imposes the participation of appropriately charged species (ions) in the protein binding mechanism. Although, there have been several qualitative studies on the promotion of binding of anionic biomolecules such as DNA to negatively charged solids such as mica, 12−15 or other solids, 16 there have been no quantitative studies evaluating the role of metal ions in biomolecule binding to ionic solids. Previously, protein binding to charged solid surfaces was proposed to involve the sequestration or release of ions at/from the protein−solid interface. 17,18 That is, binding of negatively charged proteins to negatively charged solid would require sequestration of cations of proper charge, affinity and concentration to support protein binding. 17,18 The ion sequestration at the interface would neutralize the excess charge and facilitate protein assembly, and this ion-coupled protein binding (ICPB) model, where the metal ions played a critical role in protein binding, was also supported by pH and temperature dependence studies. 17 Here, the ICPB model is tested explicitly, and we demonstrate metal-mediated binding of two model proteins glucose oxidase (GO) and met-hemoglobin (Hb) to anionic α- zirconium(IV) phosphate (Zr(HPO 4 ) 2 ·H 2 O, abbreviated as α- ZrP). 19,20 α-ZrP consists of chemically and topologically homogeneous nanosheets, with large surface area per unit mass and high charge density. The stacks of α-ZrP nanosheets Received: July 30, 2012 Revised: February 1, 2013 Published: February 1, 2013 Article pubs.acs.org/Langmuir © 2013 American Chemical Society 2971 dx.doi.org/10.1021/la304979s | Langmuir 2013, 29, 2971−2981