Abstract
The metallopeptidases thimet oligopeptidase (THOP, EC 3.4.24.25) and neurolysin (NEL, EC 3.4.24.26) are enzymes that belong to the zinc endopeptidase M13 family. Numerous studies suggest that these peptidases participate in the processing of bioactive peptides such as angiotensins and bradykinin. Efforts have been conducted to develop biotechnological tools to make possible the use of both proteases to regulate blood pressure in mice, mainly limited by the low plasmatic stability of the enzymes. In the present study, it was investigated the use of nanotechnology as an efficient strategy for to circumvent the low stability of the proteases. Recombinant THOP and NEL were immobilized in gold nanoparticles (GNPs) synthesized in situ using HEPES and the enzymes as reducing and stabilizing agents. The formation of rTHOP-GNP and rNEL-GNP was characterized by the surface plasmon resonance band, zeta potential and atomic force microscopy. The gain of structural stability and activity of rTHOP and rNEL immobilized on GNPs was demonstrated by assays using fluorogenic substrates. The enzymes were also efficiently immobilized on GNPs fabricated with sodium borohydride. The efficient immobilization of the oligopeptidases in gold nanoparticles with gain of stability may facilitate the use of the enzymes in therapies related to pressure regulation and stroke, and as a tool for studying the physiological and pathological roles of both proteases.
Highlights
Gold nanoparticles (GNPs) are attractive for biological applications because of the stability, biocompatibility, facile functionalization and compatibility with organic molecules containing thiol groups [1]
A diversity of biomolecules and their mimetics has been used for in-situ synthesis of GNPs, but the use of proteins is attractive because these biomolecules act as a template and reducing agents for GNP fabrication [10,11,12,13]
Several enzymes have the potential for therapeutic applications that can be limited by their stability, and this is the case of the enzymes thimet oligopeptidase (THOP; EC 3.4.24.15) and neurolysin (NEL; EC 3.4.24.16)
Summary
Gold nanoparticles (GNPs) are attractive for biological applications because of the stability, biocompatibility, facile functionalization and compatibility with organic molecules containing thiol groups [1]. GNPs can be synthesized by one-pot, rapid processes, including the in-situ synthesis using the organic functionalizing agent as the reducing compound to convert gold ions (Au3+ ) to the metallic form [7,8,9]. A diversity of biomolecules and their mimetics has been used for in-situ synthesis of GNPs, but the use of proteins is attractive because these biomolecules act as a template and reducing agents for GNP fabrication [10,11,12,13]. Several enzymes have the potential for therapeutic applications that can be limited by their stability, and this is the case of the enzymes thimet oligopeptidase (THOP; EC 3.4.24.15) and neurolysin (NEL; EC 3.4.24.16). THOP and NEL promise as hypotensive therapeutics despite their low structural
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