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.

Electrostatic forces could contribute significantly toward enzyme−solid interactions, and controlling these charge−charge interactions while maintaining high affinity, benign adsorption of enzymes on solids is a challenge. Here, we demonstrate that chemical modification of the surface carboxyl groups of enzymes can be used to adjust the net charge of the enzyme and control binding affinities to solid surfaces. Negatively charged nanosolid, α-Zr(HPO 4 ) 2 •H 2 O (abbreviated as α-ZrP) and two negatively charged proteins, glucose oxidase (GO) and methemoglobin (Hb), have been chosen as model systems. A limited number of the aspartate and glutamate side chains of these proteins are covalently modified with tetraethylenepentamine (TEPA) to convert these negatively charged proteins into the corresponding positively charged ones (cationized). Cationized proteins retained their structure and activities to a significant extent, and the influence of cationization on binding affinities has been tested. Cationized GO, for example, showed 250-fold increase in affinity for the negatively charged α-ZrP, when compared to that of the unmodified GO, and cationized Hb, similarly, indicated 26-fold increase in affinity. Circular dichroism spectra showed that α-ZrP-bound cationized GO retained native-like structure to a significant extent, and activity studies showed that cationized GO/α-ZrP complex is ∼2.5-fold more active than GO/α-ZrP. Cationized Hb/α-ZrP retained ∼75% of activity of Hb/α-ZrP. Therefore, enzyme cationization enhanced affinities by 1−2 orders of magnitude, while retaining considerable activity for the bound biocatalyst. This benign, chemical control over enzyme charge provided a powerful new strategy to rationally modulate enzyme−solid interactions while retaining their biocatalytic properties.

The proteasome, a validated anticancer target, participates in an array of biochemical activities, which range from the proteolysis of defective proteins to antigen presentation. We report the preparation of biochemically and photophysically distinct green, red, and far-red real-time sensors designed to simultaneously monitor the proteasome’s chymotrypsin-, trypsin-, and caspase-like activities, respectively. These sensors were employed to assess the effect of simultaneous multiple active site catalysis on the kinetic properties of the individual subunits. Furthermore, we have found that the catalytic signature of the proteasome varies depending on the source, cell type, and disease state. Trypsin-like activity is more pronounced in yeast than in mammals, whereas chymotrypsin-like activity is the only activity detectable in B-cells (unlike other mammalian cells). Furthermore, chymotrypsin-like activity is more prominent in transformed B cells relative to their counterparts from healthy donors.

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