Biophysical studies of activity, stability and conformational changes in proteins: Phospholipase A2 and azurin

This Dissertation presents the work performed in two projects. The objective of the first project was to the study how the immobilization of phospholipase A2 (PLA2) from Agkistrodon piscivorus piscivorus affects its enzymatic activity. The interaction with substrates and co factor. The enzyme was immobilized in a porous silica monolith prepared using sol-gel technology. Performing enzymatic assays using 4-nitro-3-octonoyloxy benzoic acid (NOB) as substrate we demonstrated that PLA2 retained its activity after the immobilization. The enzymatic activity and its dependence on its co-factor, calcium, allowed to study the diffusion properties of substrates through the pores of the solid matrix. With kinetic studies it was possible to monitor the dependence of the activity of the immobilized enzyme on its co-factor. Reaction rates of the entrapped PLA2 presented a dependence on calcium concentration that is similar to that of the free enzyme. The retention of the enzymatic activity confirmed that the confinement in the pores of the network, does not affect the acquisition of the active conformation of the enzyme.; In addition, gadolinium (III) was studied as a possible co-factor of PLA2. The interaction of gadolinium with the enzyme was studied monitoring the fluorescence of tryptophans of PLA2. Our results indicated that Gd3+ binds in the active site of the enzyme, although no activity of the enzyme was observed in the presence of this ion.; The objective of the second project was the study of conformational changes of the protein azurin from Pseudomona aeruginosa. Changes in the secondary and tertiary structure of azurin were induced through chemical denaturation protocols. Changes in the conformation of azurin were monitored using spectroscopic techniques due to the presence of two intrinsic probes on its structure. The interaction present between Cu2+ with Cys 112 in the metal site of the protein allowed monitoring changes in this specific site of the protein. The presence of only one tryptophan in the structure of azurin served as a fluorescent probe to monitor changes on its tertiary structure during the denaturation process. Conformational changes of azurin were studied with different approaches such as the addition of a co-solvent to stabilize the structure, the immobilization of azurin in sol-gel materials to alter solvation properties and replacing Cu2+ for Gd 3+. The results obtained from these experiments gave information about changes on the secondary and tertiary structure of the protein during the denaturation process from different points of view. It was possible to identify the presence of an intermediary structure during the chemical denaturation process exclusively in the presence of copper.