Identificação e caracterização de inibidor de protease no extrato de glândula do veneno de Polybia Paulista

Abstract

The poison of the social wasp Polybia paulista, usually formed of a complex mixture of components, proves to be a great area of study for the discovery of new biomolecules. Protease inhibitors are proteins or peptides widely distributed in nature; are molecules that have the ability to inhibit the action of proteolytic enzymes forming stable complexes with target proteases.Investigating the biological and pharmacological properties of protease inhibitors is essential to understand their mechanisms in pathophysiological processes, in addition to their potential use for economic and therapeutic purposes. The objective of the work was to identification and characterization of protease inhibitors in wasp venom gland extract. For this, the proteins from the poison were first extracted by manual dissection and stored in PBS. Afterwards, the presence of the protease inhibitor was identified by reverse zymography copolymerized with gelatin, incubated with trypsin. Then, the protease inhibitor was characterized by reverse zymography, using different substrates in conjunction with the incubation of different proteolytic enzymes: trypsin, papain, α-chymotrypsin, proteinase k and pepsin. After this process, the protein components of the poison extract of Polybia paulista were enriched by means of molecular exclusion chromatography using the Yara Phenomenex Sec. 2000 column. Thus, it was possible to trace the elutions to find the protease inhibitor of the chromatographic fractions, using SDS-PAGE gradient (20 to 5%) stained with monochromatic silver staining and reverse zymography at different incubation times stained with Comassie blue R -250.The presence of protease inhibitor on trypsin was identified in the two nests used for this work, both with electrophoretic migration close to 150kDa.The possible interaction of the reducing agent β-mercaptoethanol with temperature was investigated, loss of inhibitory activity was observed at 100 ° C. and the non-interaction of the reducing agent with the inhibitor present in the extract. In the characterization with the enzymes and substrates, an inhibitor with 150 kDa was visualized with the gelatin substrate that inhibited the enzymes trypsin, α-chymotrypsin and proteinase K, all belonging to the class of serine proteases. In the casein substrate an inhibitor with 100 kDa was found and with the substrates ovoalbumin and BSA an inhibitor of 60kDa.With the enzyme papain of the class of cysteine protease inhibitors it showed the same electrophoretic migration that was found in all other enzymes in their respective substrates. It was possible to evaluate the optimal pH of inhibitory activity with the enzyme pepsin that requires a pH 2.0 for its activity, showing that the inhibitors are influenced by pH. Molecular exclusion chromatography allowed the molecules to separate. Considering the results obtained, the protease inhibitors present in the extract of the venom gland of P. paulista collaborate with other researchers who show the presence of inhibitors of serine-cysteine protease in the venom, there are still few understood in their functions that would need to be answered and elucidated.