Kinetic characterization of gyroxin, a serine protease from Crotalus durissus terrificus venom

Abstract

This work describes for the first time the characterization of the enzymatic features of gyroxin, a serine protease from Crotalus durissus terrificus venom, capable to induce barrel rotation syndrome in rodents. Measuring the hydrolysis of the substrate ZFR-MCA, the optimal pH for proteolytic cleavage of gyroxin was found to be at pH 8.4. Increases in the hydrolytic activity were observed at temperatures from 25 °C to 45 °C, and increases of NaCl concentration up to 1 M led to activity decreases. The preference of gyroxin for Arg residues at the substrate P1 position was also demonstrated. Taken together, this work describes the characterization of substrate specificity of gyroxin, as well as the effects of salt and pH on its enzymatic activity.

Edema Induced by a Crotalus durissus terrificus Venom Serine Protease (Cdtsp 2) Involves the PAR Pathway and PKC and PLC Activation

“…In summary, we observed that, under our experimental conditions, acute edema induced by Cdtsp 2 involves a G-protein signaling pathway coupled to PAR-type receptors. A previous study showed that the administration of specific concentrations of PAR agonists may not only decrease inflammation, but also induce hyperalgesia [42]. Lastly, our results clearly suggest that edema induced by Cdtsp 2 leads to increased oxidative stress, as shown by the increase in levels of MDA.…”

Do Antibiotics Potentiate Proteases in Hemotoxic Snake Venoms?

“…Interestingly, in the fluorescence assay with Crotalus adamanteus (eastern diamondback rattlesnake) venom, all four antibiotics appear to potentiate cleavage at most concentrations (Figure 2A-D), with the exception of two or three of the lowest concentrations of cloxacillin and ampiclox, where the level of relative fluorescence units per minute (RFU/min) was similar to the positive control ( Figure 2C,D). All tested concentrations of kanamycin and ampiclox significantly potentiated the purified snake venom serine protease gyroxin [5] (Figure 2E,H), and so did all concentrations of ampicillin, except the second lowest concentration (0.128 µg/mL), which instead showed slightly inhibiting effects ( Figure 2F). The last antibiotic, cloxacillin, showed very interesting results on gyroxin ( Figure 2G).…”

“…The second assay employed was a quenched fluorescent peptide-based substrate assay, which monitors the cleavage of a fluorogenic peptide substrate (Cat#ES011, R&D systems, Minneapolis, MI, USA) that is a known cleavage substrate for different serine proteases involved in the blood coagulation cascade, such as thrombin (Factor II), Factor VII, and a range of kallikreins. Different concentrations of antibiotics were mixed with either 8 µg/mL C. adamanteus whole venom, 8 µg/mL B. asper whole venom, or with 6 µg/mL of the C. durissus terrificus (South American rattlesnake) serine protease, gyroxin [5]. Gyroxin was purified according to Barros et al [9].…”

Subproteome of Lachesis muta rhombeata venom and preliminary studies on LmrSP-4, a novel snake venom serine proteinase

“…Substrate hydrolysis decreased at 60 °C showing the beginning of a denaturation process and loss of activity. SVSPs are usually resistant to high temperatures [85-87] and this may be due to their sugar content and three-dimensional structure stabilized by the six conserved disulfide bonds [70].…”