Proteins Immobilized at the Galleries of Layered α-Zirconium Phosphate:  Structure and Activity Studies (2000)

Facile immobilization of myoglobin (Mb), lysozyme (Lys), hemoglobin (Hb), chymotrypsin (CT), and glucose oxidase (GO) at the interlayer regions of layered α-zirconium phosphate (α-ZrP) under ambient conditions (pH 7.2, room temperature) is described. The proteins retain their structure and activity after immobilization. The interlayer spacings of α-ZrP (observed in powder XRD experiments) increased from 7.6 Å (for α-ZrP) to 53, 47, 66, 62, and 108 Å when Mb, Lys, Hb, CT, and GO are bound to the matrix, respectively. These XRD data strongly suggest intercalation of the proteins in the galleries. The binding constants of the above proteins with α-ZrP, estimated from the centrifugation method, are in the range of 104−106 M-1. The α, β, and the soret absorption bands of Mb/Hb immobilized on α-ZrP were essentially superimposable with those of the native proteins in solution. The FTIR spectra of the enzyme−α-ZrP composites, measured with an attenuated total reflectance accessory, show that the amide I and amide II bands of the immobilized proteins correlate well with those of the free proteins. The circular dichroism (CD) spectra of immobilized proteins, similarly, are quite similar to those of the corresponding native proteins. These various spectral investigations are consistent with high affinity binding of the proteins to α-ZrP with considerable retention of protein structure. Activities of the immobilized proteins are investigated for comparison with those of the proteins in solution. The hydrolytic activity of immobilized lysozyme is essentially the same as that of the free protein. CT and GO showed small but reproducible increases in their activities upon immobilization. The enzymatic activity parameters, Km and Vmax, observed for the immobilized Mb, lysozyme, and GO are comparable to those of the native proteins. The peroxidase activity of Mb/Hb depended on the substrate structure. Activities are higher for the immobilized Mb when p-methoxyphenol, phenol, and m-aminophenol are used as the substrates. The activities have been lower, on the other hand, when aniline, o-cresol, and o-aminophenol are used as the substrates. In general, the ratio of the specific activities of immobilized Mb to those of free Mb roughly increases with increased substrate oxidation potential. The interlayer spacings observed for the protein−α-ZrP composites show a strong correlation with the average size of the protein, and this method may be useful for the determination of protein size. The binding affinities of the proteins correlate with their respective pI values, with the exception of Hb, suggesting the role of electrostatic interactions in the binding process. The average area occupied per protein molecule is larger than the average area of cross section of the proteins, and the area occupied correlates with the reciprocal of the corresponding binding constants. These studies indicate that the hydrophilic character of the matrix and its surface charge are important attributes that influence the bound protein behavior. Enhanced activity of Mb with specific substrates and improved activities of CT and GO are additional advantages of immobilization. Control over the immobilized protein properties is important for their application in biosensors and biocatalysis.