Fibrin(ogen)olytic and antiplatelet activities of a subtilisin-like protease from Solanum tuberosum (StSBTc-3)

Abstract

a b s t r a c tPlant serine proteases have been widely used in food science and technology as well as in medicine. In this sense, several plant serine proteases have been proposed as potential anti-coagulants and antiplatelet agents. Previously, we have reported the purification and identification of a plant serine protease from Solanum tuberosum leaves. This potato enzyme, named as StSBTc-3, has a molecular weight of 72 kDa and it was characterized as a subtilisin like protease. In this work we determine and characterize the biochemical and medicinal properties of StSBTc-3. Results obtained show that, like the reported to other plant serine proteases, StSBTc-3 is able to degrade all chains of human fibrinogen and to produces fibrin clot lysis in a dose dependent manner. The enzyme efficiently hydrolyzes b subunit followed by partially hydrolyzed a and g subunits of human fibrinogen. Assays performed to determine StSBTc-3 substrate specificity using oxidized insulin b-chain as substrate, show seven cleavage sites: Asn3-Gln4; Cys7-Gly8; Glu13-Ala14; Leu15-Tyr16; Tyr16-Leu17; Arg22-Gly23 and Phe25-Tyr26, all of them were previously reported for other serine proteases with fibrinogenolytic activity. The maximum StSBTc-3 fibrinogenolytic activity was determined at pH 8.0 and at 37 C. Additionally, we demonstrate that StSBTc-3 is able to inhibit platelet aggregation and is unable to exert cytotoxic activity on human erythrocytes in vitro at all concentrations assayed. These results suggest that StSBTc-3 could be evaluated as a new agent to be used in the treatment of thromboembolic disorders such as strokes, pulmonary embolism and deep vein thrombosis.

Optimization of fibrinogenolytic activity of Solanum tuberosum subtilisin-like protease (StSBTc-3) by response surface methodology

“…The eluted fraction with protease activity where purified again, through size exclusion chromatography to obtain the pure enzyme. The detailed protocol for purification and identification of this protease can be found elsewhere [8] , [7] .…”

“…Responses were evaluated measuring the fibrinogenolytic activity of St SBTc-3. In order to do that the method utilized by Pepe et al [8] was used. Solutions containing 40 μg of human fibrinogen were incubated with St SBTc-3.…”

“…We have already reported the isolation and partial biochemical characterization of St STBc-3, a subtilisin-like protease from Solanum tuberosum leaves [7] . Data obtained demonstrate that St STBc-3 degrades all chains of human fibrinogen in vitro and inhibits platelet aggregation induced by collagen; therefore, St STBc-3 has potential to be used in therapeutics for cardiovascular diseases [8] .…”

“…Several factors like pH, temperature and enzyme concentration affect enzymatic activities. In a previous work we carried out a preliminary study on the effect that these variables have on the fibrinogenolytic activity [8] . However, this approach did not take into account the possible mutual interactions between variables.…”

Gene Clone and Bioinformatics Analysis of Subtilisin-Like Protease in Cucumis sativus

“…SUBP is a serine protease widely found in plants, bacteria, fungi, and parasites [3][4][5][6][7][8]. Such proteases have a typical Asp/Ser/His catalytic domain in structure.…”