Application): The fundamental concept of the proposed research is to investigate the factors that affect reactions catalyzed by enzymes immobilized in sol-gel glasses. Although there is an immense number of proteins that perform important and well-documented physiological functions, astonishingly little use has been made of their therapeutic properties, the reason being in many cases that they are sensitive and chemically unstable. In particular, proteins entrapped in sol-gel materials can be used to develop biosensors for the detection of metabolites, thereby being promising for the development of novel clinical analytical methods. Phospholipases A2 (PLA2) represent ideal models to study enzymatic reactions in porous media because these proteins are small, rigid and stable, and catalyze an elementary class of reactions (hydrolysis). The investigators immobilized Agkistrodom piscivorus piscivorus phospholipase A2 in silica sol-gels and have observed the hydrolysis of 3-octonoyloxy-4-nitrobenzoic acid (ONB), following the appearance in the visible region of the nitrophenolate ion. This hydrolysis reaction will be carried out to test changes in activity, considering experimental factors such as aging/structural evolution, storage conditions, temperature, drying, polarity, pH, the accessibility of different substrates, concentration of different divalent metals, and conformational states of the enzyme. To understand the diffusion properties of molecules and ions through the interstices of the silica sol-gel is an aspect that they plan to address. They have entrapped organic fluorescent dyes and have observed the attenuation of the fluorescence signal as a function of time when the monoliths are put in contact with solutions of a quencher such as acrylamide. Since PLA2s are calcium-dependent enzymes, the diffusion properties of divalent metals such as Ca2+, Mg2+ and Zn2+ will be studied entrapping metal ion fluorescent probes. The conformational states of the protein will be assessed from fluorometric measurements. The use of quenchers such as O2, I-, and acrylamide will provide information about the accessibility of the different aromatic amino acids and about the ternary structure of the protein. The maxima of the fluorescence spectra along with the full width at half maximum of the spectra will be used to correlate different groups of tryptophans. Fluorescence lifetime measurements will also be employed to identify different tryptophil residues.
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