Purification and characterization of peptidases from the venom of Tityus serrulatus scorpion.

O escorpião amarelo é uma das principais espécies de interesse médico no Brasil e o tratamento recomendado em caso de acidentes é o uso do antiveneno. Pouco se sabe sobre os componentes proteolíticos de seu veneno e seus efeitos no envenenamento. O objetivo deste trabalho foi isolar peptidases do veneno de T. serrulatus e as caracterizar bioquimicamente, além de avaliar o potencial neutralizante de antivenenos. O veneno total foi caracterizado, buscando atividades proteolíticas relevantes. Após isso, foram isoladas duas metalloserrulases (ms3 e ms4) e uma ACE-like do veneno. Estudos bioquímicos como determinação de temperatura e pHs ótimos, influência de sais, determinação de constantes catalíticas e pontos de hidrólise dos substratos foram determinados para as proteases purificadas. Conclui-se que ativação/inativação de peptídeos bioativos in vitro pelas proteases são informações importantes e que deverão continuar sendo estudadas no futuro.The yellow scorpion is one of the main species of medical interest in Brazil and the recommended treatment in case of accidents is the use of antivenom. Little is known about the proteolytic components of its venom and its effects on envenomation. The objective of this work was to isolate peptidases from T. serrulatus venom and to characterize them biochemically, besides to evaluating the neutralizing potential of antivenoms. The total venom was characterized, in searching for relevant proteolytic activities. After that, two metalloserrulases (ms3 and ms4) and one ACE-like venom were isolated. Biochemical studies such as determination of temperature and optimum pHs, influence of salts, determination of catalytic constants and hydrolysis points of the substrates were determined for the purified proteases. It was concluded that the activation / inactivation of bioactive peptides in vitro by proteases are important information and should be further studied in the future

Purification and characterization of peptidases from the venom of Tityus serrulatus scorpion.

O escorpião amarelo é uma das principais espécies de interesse médico no Brasil e o tratamento recomendado em caso de acidentes é o uso do antiveneno. Pouco se sabe sobre os componentes proteolíticos de seu veneno e seus efeitos no envenenamento. O objetivo deste trabalho foi isolar peptidases do veneno de T. serrulatus e as caracterizar bioquimicamente, além de avaliar o potencial neutralizante de antivenenos. O veneno total foi caracterizado, buscando atividades proteolíticas relevantes. Após isso, foram isoladas duas metalloserrulases (ms3 e ms4) e uma ACE-like do veneno. Estudos bioquímicos como determinação de temperatura e pHs ótimos, influência de sais, determinação de constantes catalíticas e pontos de hidrólise dos substratos foram determinados para as proteases purificadas. Conclui-se que ativação/inativação de peptídeos bioativos in vitro pelas proteases são informações importantes e que deverão continuar sendo estudadas no futuro.The yellow scorpion is one of the main species of medical interest in Brazil and the recommended treatment in case of accidents is the use of antivenom. Little is known about the proteolytic components of its venom and its effects on envenomation. The objective of this work was to isolate peptidases from T. serrulatus venom and to characterize them biochemically, besides to evaluating the neutralizing potential of antivenoms. The total venom was characterized, in searching for relevant proteolytic activities. After that, two metalloserrulases (ms3 and ms4) and one ACE-like venom were isolated. Biochemical studies such as determination of temperature and optimum pHs, influence of salts, determination of catalytic constants and hydrolysis points of the substrates were determined for the purified proteases. It was concluded that the activation / inactivation of bioactive peptides in vitro by proteases are important information and should be further studied in the future

New Insights into the Hypotensins from <i>Tityus serrulatus</i> Venom: Pro-Inflammatory and Vasopeptidases Modulation Activities

The Tityus serrulatus scorpion is considered the most dangerous of the Brazilian fauna due to the severe clinical manifestations in injured victims. Despite being abundant components of the venom, few linear peptides have been characterized so far, such as hypotensins. In vivo studies have demonstrated that hypotensin I (TsHpt-I) exerts hypotensive activity, with an angiotensin-converting enzyme (ACE)-independent mechanism of action. Since experiments have not yet been carried out to analyze the direct interaction of hypotensins with ACE, and to deepen the knowledge about these peptides, hypotensins I and II (TsHpt-II) were studied regarding their modulatory action over the activities of ACE and neprilysin (NEP), which are the peptidases involved in blood pressure control. Aiming to search for indications of possible pro-inflammatory action, hypotensins were also analyzed for their role in murine macrophage viability, the release of interleukins and phagocytic activity. TsHpt-I and -II were used in kinetic studies with the metallopeptidases ACE and NEP, and both hypotensins were able to increase the activity of ACE. TsHpt-I presented itself as an inhibitor of NEP, whereas TsHpt-II showed weak inhibition of the enzyme. The mechanism of inhibition of TsHpt-I in relation to NEP was defined as non-competitive, with an inhibition constant (Ki) of 4.35 μM. Concerning the analysis of cell viability and modulation of interleukin levels and phagocytic activity, BALB/c mice’s naïve macrophages were used, and an increase in TNF production in the presence of TsHpt-I and -II was observed, as well as an increase in IL-6 production in the presence of TsHpt-II only. Both hypotensins were able to increase the phagocytic activity of murine macrophages in vitro. The difference between TsHpt-I and -II is the residue at position 15, with a glutamine in TsHpt-I and a glutamic acid in TsHpt-II. Despite this, kinetic analyzes and cell assays indicated different actions of TsHpt-I and -II. Taken together, these results suggest a new mechanism for the hypotensive effects of TsHpt-I and -II. Furthermore, the release of some interleukins also suggests a role for these peptides in the venom inflammatory response. Even though these molecules have been well studied, the present results suggest a new mechanism for the hypotensive effects of TsHpt-

Purification and Biochemical Characterization of TsMS 3 and TsMS 4: Neuropeptide-Degrading Metallopeptidases in the <i>Tityus serrulatus</i> Venom

Although omics studies have indicated presence of proteases on the Tityus serrulatus venom (TsV), little is known about the function of these molecules. The TsV contains metalloproteases that cleave a series of human neuropeptides, including the dynorphin A (1-13) and the members of neuropeptide Y family. Aiming to isolate the proteases responsible for this activity, the metalloserrulase 3 and 4 (TsMS 3 and TsMS 4) were purified after two chromatographic steps and identified by mass spectrometry analysis. The biochemical parameters (pH, temperature and cation effects) were determined for both proteases, and the catalytic parameters (Km, kcat, cleavage sites) of TsMS 4 over fluorescent substrate were obtained. The metalloserrulases have a high preference for cleaving neuropeptides but presented different primary specificities. For example, the Leu-enkephalin released from dynorphin A (1-13) hydrolysis was exclusively performed by TsMS 3. Neutralization assays using Butantan Institute antivenoms show that both metalloserrulases were well blocked. Although TsMS 3 and TsMS 4 were previously described through cDNA library studies using the venom gland, this is the first time that both these toxins were purified. Thus, this study represents a step further in understanding the mechanism of scorpion venom metalloproteases, which may act as possible neuropeptidases in the envenomation process

Tityus serrulatus Scorpion Venom: In Vitro Tests and Their Correlation with In Vivo Lethal Dose Assay

Scorpion stings are the main cause of human envenomation in Brazil and, for the treatment of victims, the World Health Organization (WHO) recommends the use of antivenoms. The first step to achieve effective antivenom is to use a good quality venom pool and to evaluate it, with LD50 determination as the most accepted procedure. It is, however, time-consuming and requires advanced technical training. Further, there are significant ethical concerns regarding the number of animals required for testing. Hence, we investigated the correspondence between LD50 results, in vitro assays, and a strong correlation with proteolytic activity levels was observed, showing, remarkably, that proteases are potential toxicity markers for Tityus serrulatus venom. The comparison of reversed-phase chromatographic profiles also has a potential application in venoms’ quality control, as there were fewer neurotoxins detected in the venom with high LD50 value. These results were confirmed by mass spectrometry analysis. Therefore, these methods could precede the LD50 assay to evaluate the venom excellence by discriminating—and discarding—poor-quality batches, and, consequently, with a positive impact on the number of animals used. Notably, proposed assays are fast and inexpensive, being technically and economically feasible in Tityus serrulatus venom quality control to produce effective antivenoms