Nanoencapsulation of cytochrome c and horseradish peroxidase at the galleries of alpha-zirconium phosphate

Cytochrome c (Cyt c) and horseradish peroxidase (HRP) are encapsulated in the galleries of a-zirconium phosphate (Zr[HPO4](2) . nH(2)O, alpha-ZrP) and alpha-zirconium phosphonate Zr(PO3-CH2COOH)(2) . nH(2)O (alpha-ZrPAA), under mild conditions (pH 7.2, room temperature). Thermal stability, peroxidase activity, and the spectral features of the bound proteins are largely preserved. The binding constants for alpha-ZrP are in the range 1-100 mu M(-1) Cyt c shows a much higher affinity for alpha-ZrP (42/mu M) than KRP (1.5/mu M) and alpha-ZrP has a much higher affinity for Cyt c than alpha-ZrPAA. The binding interactions are, thus, sensitive to the surface functional groups of the protein as well as the ZrP matrix. The alpha, beta, and the soret absorption bands of heme protein-alpha-ZrP composites were essentially superimposable with those of the native protein. The FTIR spectra of the protein-phosphate composites are superimposable with those of the native proteins indicating no major changes in the secondary structures of the bound proteins. Powder diffraction data of protein/alpha-ZrP indicate increases in the interlayer spacings from 7.6 Angstrom for alpha-ZrP to similar to 33 Angstrom for protein/alpha-ZrP samples, providing strong evidence for the intercalation of proteins in the galleries. Dithionite or ascorbate reduces ferric Cyt c/alpha-ZrP to the ferrous form, while hydrogen peroxide rapidly reacts with HRP/alpha-ZrP to form the green intermediate. Ferricyanide readily oxidizes the ferrous Cyt c/alpha-ZrP to ferric Cyt c/alpha-ZrP, and alpha-ZrP bound Cyt c retains its peroxidase activity. The thermal stability of the bound Cyt c is similar to that of free protein.