Binding and cleavage studies of two proteins intercalated at the galleries of α-zirconium phosphate

Met hemoglobin (Hb) and lysozyme are intercalated in the galleries of alpha-Zr(IV)phosphate (alpha-ZrP), and the resulting bioactive materials have been characterized using scanning electron microscopy, calorimetry and chemical footprinting methods. Microscopy revealed that the inorganic material crystallizes as nanoscopic discs of 600 nm in diameter, and that the layered structure is retained when proteins are intercalated in the galleries. Isothermal titration calorimetric studies show that binding of Hb to alpha-ZrP at 25 degreesC is strongly exothermic (DeltaH = 31.0 +/- 2.5 kcal/mol) with a decrease in entropy (DeltaS = -77 +/- 8 e.u.), while a model system such as Mg(II) binding to alpha-ZrP was endothermic (DeltaH(1) = 2.4 +/- 0.03 kcal/mol, DeltaH(2) = 4.3 +/- 0.3 kcal/mol). These differences in the binding thermodynamics of the two guests clearly indicate that a simple electrostatic model for the binding process is not adequate to explain Hb binding to a-ZrP. The protein-solid composites are further characterized in chemical footprinting studies. PolyammineCo(III) complexes cleave Hb and lysozyme at specific sites, and these have been used as probes to examine protein-solid contact regions. Protein cleavage studies indicated that Hb bound to a-ZrP is cleaved at four specific locations, and that these sites are accessible to the Co(III) reagents. Such access strongly suggests that the Hb tetramer is oriented in the galleries with its long axis parallel to the galleries. This conclusion is consistent with the powder X-ray diffraction (XRD) and activity studies reported earlier. Most likely this is one of the significant orientations of Hb at the galleries while other orientations are also possible. Similar footprinting studies with lysozyme bound in the galleries indicated that lysozyme is cleaved predominantly at a single site, in good yield (28%). Current results support the idea that chemical footprinting methods, when coupled with powder XRD and activity studies, are useful to examine specific orientations of the proteins at solid surfaces. (C) 2004 Elsevier Inc. All rights reserved.