Measurement of Fluorescence from Tryptophan To Probe the Environment and Reaction Kinetics within Protein-Doped Sol−Gel-Derived Glass Monoliths

Abstract

Optically clear, ultrathin monoliths that contained the single-tryptophan protein monellin were prepared by the sol-gel technique from tetraethyl orthosilicate (TEOS). Suitable precautions were established to eliminate background fluorescence from impurities in TEOS, scattering from the monoliths, and photobleaching of the entrapped protein. Fluorescence spectra and anisotropy results indicated that useful, essentially scatter-free fluorescence signals could be obtained from the intrinsic tryptophan residue of monellin which was entrapped into either wet-aged or dry-aged monoliths. The combination of spectral, quenching, and anisotropy results suggested that the mobility of solvent inside monoliths was substantially reduced compared to bulk solution, providing a possible explanation for the improvements in protein stability that occur upon entrapment. The monitoring of intrinsic protein fluorescence also provided information about the kinetics of the interaction between the entrapped protein and external reagents. The interaction of monellin with both neutral and charged species was examined under conditions of continuous stirring and indicated response times on the order of minutes. In the case of the neutral species, the kinetics were best described by a sum of first-order rate constants when the reactions occurred in the glass matrix. For charged species, interactions between the analytes and the negatively charged glass matrix caused the reaction kinetics to become complex, with the overall reaction rate depending on both the type of aging and the charge on the analyte. These findings suggest that caution must be exercised when entrapped proteins are used for sensing of charged species.

Conformational Changes in Azurin from Pseudomona aeruginosa Induced through Chemical and Physical Protocols

“…Using this procedure we observed a marked increased in the stability of the protein (data not shown) ( f D ¼ 0.5 at [GuHCl] % 4 M) as compared to the protein immobilized using Brenan's protocol (Fig. 2 B) (Zheng et al, 1997). However, the stability of the protein immobilized using Friedman's method is comparable to that of the protein free in solution, DG 0 w ¼ ÿ42:0 kJ=mol: As a control experiment, we carried out a denaturation experiment for the protein free in solution, in the presence of 15% glycerol.…”

“…Sol-gel silica monoliths were prepared following the procedure described by Brenan and co-workers (Zheng et al, 1997). To initiate the hydrolysis of TEOS an acidified aqueous solution was added, but the formation of a new phase was observed.…”

Biosensors Based on Sol–Gel-Derived Materials

“…37,38 Entrapment can also stabilize proteins against other denaturing forces, for example, in the case of catalase, the silica gel stabilises the encapsulated enzyme to higher methanol concentrations. Oxidation of either HRP or Mb by peroxide can also be minimized by immobilization of the proteins in the silicate matrix.…”

Organosilicon Biotechnology

“…Typically the sol-gel process is initiated by combining acidic or alkaline water: alcohol mixtures with tetra-or tri-alkoxysilanes, followed by ageing of the sol-gel to form a gelatinous matrix. Several approaches have been designed to control the physical characteristics of the final gel, including combination acid/ base catalysis [94], and solvent free methods [95].…”