Kinetic investigations and stability studies of two Bothrops L-amino acid oxidases

L-amino acid oxidases isolated from snake venoms (SV-LAAOs) are enzymes that have great therapeutic potential and are currently being investigated as tools for developing new strategies to treat various diseases, including cancer and bacterial infections. The main objective of this study was to make a brief evaluation of the enzymatic stability of two Bothrops LAAOs, one isolated from Bothrops jararacussu (BjussuLAAO-II) and the other from Bothrops moojeni (BmooLAAO-I) venoms. Methods and results: The enzymatic activity and stability of both LAAOs were evaluated by microplate colorimetric assays, for which BjussuLAAO-II and BmooLAAO-I were incubated with different L-amino acid substrates, in the presence of different ions, and at different pH ranges and temperatures. BjussuLAAO-II and BmooLAAO-I demonstrated higher affinity for hydrophobic amino acids, such as Phe and Leu. The two enzymes showed high enzymatic activity in a wide temperature range, from 25 to 75 °C, and presented optimum pH around 7.0. Additionally, Zn2+, Al3+, Cu2+ and Ni2+ ions negatively modulated the enzymatic activity of both LAAOs. As to stability, BjussuLAAO-II and BmooLAAO-I showed high enzymatic activity for 42 days stored at 4°C in neutral pH solution. Moreover, the glycan portions of both LAAOs were analyzed by capillary electrophoresis, which revealed that BjussuLAAO-II presented two main glycan portions with relative masses of 7.78 and 8.13 CGU, while BmooLAAO-I showed three portions of 7.58, 7.94 and 8.37 CGU. Conclusions: Our results showed that, when stored properly, BjussuLAAO-II and BmooLAAO-I present enzymatic stability over a long time period, which is very important to allow the use of these enzymes in pharmacological studies of great impact in the medical field.(AU)

Cytotoxic and inflammatory potential of a phospholipase A2 from Bothrops jararaca snake venom

Snake venom phospholipases A2 (PLA2s) have been reported to induce myotoxic, neurotoxic, hemolytic, edematogenic, cytotoxic and proinflammatory effects. This work aimed at the isolation and functional characterization of a PLA2 isolated from Bothrops jararaca venom, named BJ-PLA2-I. Methods and Results: For its purification, three consecutive chromatographic steps were used (Sephacryl S-200, Source 15Q and Mono Q 5/50 GL). BJ-PLA2-I showed acidic characteristics, with pI~4.4 and molecular mass of 14. 2 kDa. Sequencing resulted in 60 amino acid residues that showed high similarity to other Bothrops PLA2s, including 100% identity with BJ-PLA2, an Asp49 PLA2 previously isolated from B. jararaca venom. Being an Asp49 PLA2, BJ-PLA2-I showed high catalytic activity, and also inhibitory effects on the ADP-induced platelet aggregation. Its inflammatory characterization showed that BJ-PLA2-I was able to promote leukocyte migration in mice at different concentrations (5, 10 and 20 µg/mL) and also at different response periods (2, 4 and 24 h), mainly by stimulating neutrophil infiltration. Furthermore, increased levels of total proteins, IL-6, IL-1 ß and PGE2 were observed in the inflammatory exudate induced by BJ-PLA2-I, while nitric oxide, TNF-α, IL-10 and LTB4 levels were not significantly altered. This toxin was also evaluated for its cytotoxic potential on normal (PBMC) and tumor cell lines (HL-60 and HepG2). Overall, BJ-PLA2-I (2.5-160 µg/mL) promoted low cytotoxicity, with cell viabilities mostly varying between 70 and 80% and significant values obtained for HL-60 and PBMC only at the highest concentrations of the toxin evaluated. Conclusions: BJ-PLA2-I was characterized as an acidic Asp49 PLA2 that induces acute local inflammation and low cytotoxicity. These results should contribute to elucidate the action mechanisms of snake venom PLA2s.(AU)

Antitumor potential of the myotoxin BthTX-I from Bothrops jararacussu snake venom: evaluation of cell cycle alterations and death mechanisms induced in tumor cell lines

Background Phospholipases A 2 (PLA 2 s) are abundant components of snake venoms that have been extensively studied due to their pharmacological and pathophysiological effects on living organisms. This study aimed to assess the antitumor potential of BthTX-I, a basic myotoxic PLA 2isolated from Bothrops jararacussu venom, by evaluating in vitro processes of cytotoxicity, modulation of the cell cycle and induction of apoptosis in human (HL-60 and HepG2) and murine (PC-12 and B16F10) tumor cell lines. Methods The cytotoxic effects of BthTX-I were evaluated on the tumor cell lines HL-60 (promyelocytic leukemia), HepG2 (human hepatocellular carcinoma), PC-12 (murine pheochromocytoma) and B16F10 (murine melanoma) using the MTT method. Flow cytometry technique was used for the analysis of cell cycle alterations and death mechanisms (apoptosis and/or necrosis) induced in tumor cells after treatment with BthTX-I. Results It was observed that BthTX-I was cytotoxic to all evaluated tumor cell lines, reducing their viability in 40 to 50 %. The myotoxin showed modulating effects on the cell cycle of PC-12 and B16F10 cells, promoting delay in the G0/G1 phase. Additionally, flow cytometry analysis indicated cell death mainly by apoptosis. B16F10 was more susceptible to the effects of BthTX-I, with ~40 % of the cells analyzed in apoptosis, followed by HepG2 (~35 %), PC-12 (~25 %) and HL-60 (~4 %). Conclusions These results suggest that BthTX-I presents antitumor properties that may be useful for developing new therapeutic strategies against cancer.(AU)

Purification procedure for the isolation of a P-I metalloprotease and an acidic phospholipase A 2 fromBothrops atrox snake venom

Background Snake venoms are complex mixtures of inorganic and organic components, mainly proteins and peptides. Standardization of methods for isolating bioactive molecules from snake venoms is extremely difficult due to the complex and highly variable composition of venoms, which can be influenced by factors such as age and geographic location of the specimen. Therefore, this study aimed to standardize a simple purification methodology for obtaining a P-I class metalloprotease (MP) and an acidic phospholipase A2 (PLA 2 ) from Bothrops atroxvenom, and biochemically characterize these molecules to enable future functional studies.Methods To obtain the toxins of interest, a method has been standardized using consecutive isolation steps. The purity level of the molecules was confirmed by RP-HPLC and SDS-PAGE. The enzymes were characterized by determining their molecular masses, isoelectric points, specific functional activity and partial amino acid sequencing.Results The metalloprotease presented molecular mass of 22.9 kDa and pI 7.4, with hemorrhagic and fibrin(ogen)olytic activities, and its partial amino acid sequence revealed high similarity with other P-I class metalloproteases. These results suggest that the isolated metalloprotease is Batroxase, a P-I metalloprotease previously described by our research group. The phospholipase A 2 showed molecular mass of 13.7 kDa and pI 6.5, with high phospholipase activity and similarity to other acidic PLA2 s from snake venoms. These data suggest that the acidic PLA2 is a novel enzyme from B. atrox venom, being denominated BatroxPLA 2 .Conclusions The present study successfully standardized a simple methodology to isolate the metalloprotease Batroxase and the acidic PLA 2 BatroxPLA2 from the venom of B. atrox, consisting mainly of classical chromatographic processes. These two enzymes will be used in future studies to evaluate their effects on the complement system and the inflammatory process, in addition to the thrombolytic potential of the metalloprotease.(AU)

Purification procedure for the isolation of a P-I metalloprotease and an acidic phospholipase A2 from Bothrops atrox snake venom

Background Snake venoms are complex mixtures of inorganic and organic components, mainly proteins and peptides. Standardization of methods for isolating bioactive molecules from snake venoms is extremely difficult due to the complex and highly variable composition of venoms, which can be influenced by factors such as age and geographic location of the specimen. Therefore, this study aimed to standardize a simple purification methodology for obtaining a P-I class metalloprotease (MP) and an acidic phospholipase A2 (PLA 2 ) from Bothrops atroxvenom, and biochemically characterize these molecules to enable future functional studies.Methods To obtain the toxins of interest, a method has been standardized using consecutive isolation steps. The purity level of the molecules was confirmed by RP-HPLC and SDS-PAGE. The enzymes were characterized by determining their molecular masses, isoelectric points, specific functional activity and partial amino acid sequencing.Results The metalloprotease presented molecular mass of 22.9 kDa and pI 7.4, with hemorrhagic and fibrin(ogen)olytic activities, and its partial amino acid sequence revealed high similarity with other P-I class metalloproteases. These results suggest that the isolated metalloprotease is Batroxase, a P-I metalloprotease previously described by our research group. The phospholipase A 2 showed molecular mass of 13.7 kDa and pI 6.5, with high phospholipase activity and similarity to other acidic PLA2 s from snake venoms. These data suggest that the acidic PLA2 is a novel enzyme from B. atrox venom, being denominated BatroxPLA 2 .Conclusions The present study successfully standardized a simple methodology to isolate the metalloprotease Batroxase and the acidic PLA 2 BatroxPLA2 from the venom of B. atrox, consisting mainly of classical chromatographic processes. These two enzymes will be used in future studies to evaluate their effects on the complement system and the inflammatory process, in addition to the thrombolytic potential of the metalloprotease.

Antitumor potential of the myotoxin BthTX-I from Bothrops jararacussu snake venom: evaluation of cell cycle alterations and death mechanisms induced in tumor cell lines

Background Phospholipases A 2 (PLA 2 s) are abundant components of snake venoms that have been extensively studied due to their pharmacological and pathophysiological effects on living organisms. This study aimed to assess the antitumor potential of BthTX-I, a basic myotoxic PLA 2isolated from Bothrops jararacussu venom, by evaluating in vitro processes of cytotoxicity, modulation of the cell cycle and induction of apoptosis in human (HL-60 and HepG2) and murine (PC-12 and B16F10) tumor cell lines. Methods The cytotoxic effects of BthTX-I were evaluated on the tumor cell lines HL-60 (promyelocytic leukemia), HepG2 (human hepatocellular carcinoma), PC-12 (murine pheochromocytoma) and B16F10 (murine melanoma) using the MTT method. Flow cytometry technique was used for the analysis of cell cycle alterations and death mechanisms (apoptosis and/or necrosis) induced in tumor cells after treatment with BthTX-I. Results It was observed that BthTX-I was cytotoxic to all evaluated tumor cell lines, reducing their viability in 40 to 50 %. The myotoxin showed modulating effects on the cell cycle of PC-12 and B16F10 cells, promoting delay in the G0/G1 phase. Additionally, flow cytometry analysis indicated cell death mainly by apoptosis. B16F10 was more susceptible to the effects of BthTX-I, with ~40 % of the cells analyzed in apoptosis, followed by HepG2 (~35 %), PC-12 (~25 %) and HL-60 (~4 %). Conclusions These results suggest that BthTX-I presents antitumor properties that may be useful for developing new therapeutic strategies against cancer.

Snake venom L-amino acid oxidases: an overview on their antitumor effects

The L-amino acid oxidases (LAAOs) constitute a major component of snake venoms and have been widely studied due to their widespread presence and various effects, such as apoptosis induction, cytotoxicity, induction and/or inhibition of platelet aggregation, hemorrhage, hemolysis, edema, as well as antimicrobial, antiparasitic and anti-HIV activities. The isolated and characterized snake venom LAAOs have become important research targets due to their potential biotechnological applications in pursuit for new drugs of interest in the scientific and medical fields. The current study discusses the antitumor effects of snake venom LAAOs described in the literature to date, highlighting the mechanisms of apoptosis induction proposed for this class of proteins.

Snake venom L-amino acid oxidases: an overview on their antitumor effects

The L-amino acid oxidases (LAAOs) constitute a major component of snake venoms and have been widely studied due to their widespread presence and various effects, such as apoptosis induction, cytotoxicity, induction and/or inhibition of platelet aggregation, hemorrhage, hemolysis, edema, as well as antimicrobial, antiparasitic and anti-HIV activities. The isolated and characterized snake venom LAAOs have become important research targets due to their potential biotechnological applications in pursuit for new drugs of interest in the scientific and medical fields. The current study discusses the antitumor effects of snake venom LAAOs described in the literature to date, highlighting the mechanisms of apoptosis induction proposed for this class of proteins.