Molecular cloning of albolatin, a novel snake venom metalloprotease from green pit viper (Trimeresurus albolabris), and expression of its disintegrin domain

Disintegrins are snake venom-derived, RGD- or KGD-containing peptides that can inhibit integrin-mediated platelet aggregation and cell–matix interactions. The aim of this study is to analyze the full-length cDNA sequence of a snake venom metalloprotease (SVMP) from green pit viper (Trimeresurus albolabris) venom and characterize functions of its disintegrin domain on human platelets. From the primary cDNA library of venom glands, a partial sequence of a novel SVMP (Albolatin) was obtained. Using the 5′-RACE, the 2040 bp full-length sequence of albolatin mRNA was derived. The deduced amino acid sequence revealed a type P-II SVMP of 484 amino acid residues comprising a signal region, pro-peptide, inactive metalloprotease domain and a disintegrin domain. It showed 85% amino acid identical to Trimeresurus jerdonii jerdonitin and 81% to Gloydius halys agkistin. Sequence alignment revealed that all cysteines were conserved except for an extra cysteine in the protease domain of albolatin. The disintegrin domain of albolatin, which comprised 76 amino acids with a KGDW sequence, was expressed in Pichia pastoris with the yield of 3.3 mg/L of culture medium. The molecular weights were 11 kDa in reduced and 22 kDa in non-reduced states indicating a homodimer. It can inhibit collagen-induced platelet aggregation with IC₅₀ of 976 nM and, therefore, should be investigated for a potential to be a novel therapeutic agent.     

Fractionation of snake venom metalloproteinases by metal ion affinity: A purified cobra metalloproteinase, Nk, from Naja kaouthia binds Ni-agarose

Snake venom metalloproteinases represent unique probes for analyzing platelet adhesion receptors regulating hemostasis and thrombosis. Snake venom metalloproteinase-disintegrins consist of a propeptide domain, a catalytic domain containing a metal ion-coordination sequence (HEXXHXXGXXH), a disintegrin domain, and a Cys-rich domain. Here, we investigate whether metal ion-affinity chromatography may be used to fractionate venom metalloproteinases based on the metal ion-coordination motif. First, we showed that a purified cobra metalloproteinase, Nk, from Naja kaouthia bound Ni²⁺-agarose, and was eluted by 10 mM imidazole, confirming the validity of the approach. Nk cleaved the platelet von Willebrand factor (VWF) receptor, glycoprotein (GP)Ib, with similar activity to the previously reported cobra metalloproteinase, mocarhagin, as shown by EDTA-inhibitable Nk-dependent proteolysis of a purified GPIb extracellular fragment (glycocalicin), and inhibition of ¹²⁵I-VWF binding to GPIb on washed human or canine platelets. Second, crude venom from the viper, Trimeresurus albolabris, was fractionated on Ni²⁺-agarose. Samples of flow-through, wash, and imidazole-eluted (0–30 mM gradient) fractions were analyzed by (i) SDS-polyacrylamide gel electrophoresis, (ii) immunoblotting with a rabbit anti-mocarhagin antibody, and (iii) assessing metalloproteinase activity using human fibrinogen as substrate. The combined results support the general concept of using Ni²⁺-agarose to fractionate snake venom metalloproteinases.     

Antihemorrhagin in the blood serum of king cobra (Ophiophagus hannah): purification and characterization

King cobra (Ophiophagus hannah) serum was found to possess antihemorrhagic activity against king cobra hemorrhagin. The activity was stronger than that in commercial king cobra antivenom. An antihemorrhagin has been purified by ion exchange chromatography, affinity chromatography and gel filtration with a 22-fold purification and an overall yield of 12% of the total antihemorrhagic activity contained in crude serum. The purified antihemorrhagin was homogeneous in disc-PAGE and SDS–PAGE. Its apparent molecular weight determined by SDS–PAGE was 120 kDa. The antihemorrhagin was also active against other hemorrhagic snake venoms obtained in Thailand and Japan such as Calloselasma rhodostoma, Trimeresurus albolabris, Trimeresurus macrops and Trimeresurus flavoviridis (Japanese Habu). It inhibited the proteolytic activity of king cobra venom. It is an acid- and thermolabile protein and does not form precipitin lines against king cobra venom.     

Selective anti-Toxoplasma gondii activities of azasterols

We report potent and selective inhibitory effects of 22,26-azasterol and 24,25-(R,S)-epiminolanosterol, known inhibitors of Δ²⁴⁽²⁵⁾-sterol methyltranferase (SMT) in fungi and protozoa, on the proliferation of Toxoplasma gondii in LLCMK2 cells. These compounds produced a dose-dependent reduction in parasite proliferation. 22,26-azasterol had an IC₅₀ of 5.3 μM after 24 h and 4.5 μM after 48 h, while for 24,25-(R,S)-epiminolanosterol the IC₅₀ values were 1 μM after 24 h and 0.12 μM after 48 h. The rapid reduction of parasite load suggested these compounds have selective cytotoxic effects against T. gondii. However, we were unable to detect 24-alkyl sterols in purified T. gondii tachyzoites using highly sensitive gas–liquid chromatography/mass spectrometry methods, a fact which indicated that the anti-proliferative effects of these azasterols were not mediated by inhibition of SMT. Transmission electron microscopy showed that the mitochondrion was the major target of drugs. Ultrastructural effects on plasma membrane, apicoplast and the formation of autophagosomal structures were also observed.     

Modulation of Kv4.2 channels by a peptide isolated from the venom of the giant bird-eating tarantula Theraphosa leblondi.

In order to find new peptide inhibitors for voltage-dependent potassium (Kv) channels, we examined the effects of venom from Theraphosa leblondi on Kv channel-mediated currents with the whole-cell patch-clamp technique. Both A-type currents in cultured hippocampal neurons and A-type currents recorded from HEK 293 cells transiently expressing recombinant Kv4.2 channels were selectively inhibited by T. leblondi venom. No venom activity was observed on recombinant Kv1.3, Kv1.4, Kv2.1 or Kv3.4 channels. We purified and sequenced three novel homologous peptides from this venom, which are related to previously identified Kv4 channel-specific peptide inhibitors and were named T. leblondi toxin (TLTx) 1, 2 and 3. The mode of action of TLTx1 on recombinant Kv4.2 channels was studied in more detail. TLTx1 inhibited Kv4.2-mediated currents with an IC₅₀ of 200 nM, and macroscopic current inactivation was slowed in the presence of TLTx1. Notably, TLTx1 also caused a shallower voltage dependence of Kv4.2 peak conductance and a shift of the activation midpoint to more positive potentials (ΔV_1/2=+35 mV). TLTx1 caused a noticable slowing of Kv4.2 activation kinetics, and Kv4.2 deactivation kinetics were accelerated by TLTx1 as infered from Rb⁺ tail current measurements. Chimeric Kv2.1(4.2L3-4) channels, in which the linker region between S3 and S4 of the TLTx1-insensitive Kv2.1 channel was replaced by the corresponding Kv4.2 domain, were sensitive to TLTx1. Apparently, TLTx1 can act as a gating modifier of Kv4.2 channels.     

Bothroalternin, a thrombin inhibitor from the venom of Bothrops alternatus

Bothroalternin (MW 27 kDa), a new member of the family of C-type lectins is a thrombin inhibitor which was purified from pooled B. alternatus venom by affinity chromatography on PPACK-thrombin–Sepharose, followed by size exclusion and reverse-phase on HPLC columns. Material retained on the affinity column contained proteins with apparent molecular weights ranging from 20 to 60 kDa on SDS-PAGE and inhibited aggregation of rabbit platelets induced by -thrombin ( ₅₀=28 μg/ml). A single band of 27 kDa was recognized in Western-blot assays using polyclonal antibodies raised against bothrojaracin, a thrombin inhibitor purified from B. jararaca venom (Zingali et al., 1993). The immunological similarity of this fraction to bothrojaracin was confirmed by ELISA and competitive ELISA. Further purification by size exclusion and reverse-phase on HPLC, produced a single homogenous peak called bothroalternin. This protein was highly homologous to bothrojaracin (95% in its N-terminal sequence-for residues 1 to 25) but displaying lower inhibitory effect on thrombin induced platelet aggregation ( ₅₀=0.19 μg/ml) compared to bothrojaracin ( ₅₀=0.06). Altogether, bothroalternin is a new thrombin inhibitor isolated from Bothrops alternatus venom and has been characterized as a bothrojaracin-like protein.     

Characterization of the venom from the Brazilian Brown Spider Loxosceles similis Moenkhaus, 1898 (Araneae, Sicariidae)

Accidents caused by brown spiders (Loxosceles genus) are frequent in Brazil and are associated with dermonecrotic lesions and, eventually, systemic reactions that may be lethal. The major species implicated with human envenoming have been: L. intermedia, L. gaucho and L. laeta. In this study we characterized the venom from Loxosceles similis, a species of spider normally found inside caves. L. similis venom was characterized by two-dimensional gel electrophoresis and enzymatic activity (dermonecrosis and haemolysis). The lethal dose to mice and the capacity of commercial anti-serum to neutralize this venom were also analysed. The cross-reactivity with anti-venoms against L. intermedia, L. laeta and L. gaucho were studied. Our results showed that this venom was able to induce severe dermonecrotic lesions and showed the presence of the bacteria Clostridium septicum in association with the fangs. In addition, we have cloned the DNA coding for a dermonecrotic protein (LsD1), using the genomic DNA of L. similis. The deduced amino acid sequence showed a toxin of approximately 31.2 kDa with an estimated pI of 7.37 and sequence similar to LiD1, a protein from the dermonecrotic family of Loxosceles intermedia spider venom, a synanthropic species of medical importance.     

Characterization of venom (Duvernoy’s secretion) from twelve species of colubrid snakes and partial sequence of four venom proteins

R.E. Hill and S.P. Mackessy. Characterization of venom (Duvernoy’s secretion) from twelve species of colubrid snakes and partial sequence of four venom proteins. Toxicon XX, xx–yy, 2000. — Venomous colubrids, which include more than 700 snake species worldwide, represent a vast potential source of novel biological compounds. The present study characterized venom (Duvernoy’s gland secretion) collected from twelve species of opisthoglyphous (rear-fanged) colubrid snakes, an extremely diverse assemblage of non-venomous to highly venomous snakes. Most venoms displayed proteolytic activity (casein), though activity levels varied considerably. Low phosphodiesterase activity was detected in several venoms (Amphiesma stolata, Diadophis punctatus, Heterodon nasicus kennerlyi, H. n. nasicus and Thamnophis elegans vagrans), and acetylcholinesterase was found in Boiga irregularis saliva and venom, but no venoms displayed hyaluronidase, thrombin-like or kallikrein-like activities. High phospholipase A₂ (PLA₂) activity was found in Trimorphodon biscutatus lambda venom, and moderate levels were detected in Boiga dendrophila and D. p. regalis venoms as well as B. dendrophila and H. n. nasicus salivas. Non-reducing SDS–PAGE revealed 7–20 protein bands (3.5 to over 200 kD, depending on species) for all venoms analyzed, and electrophoretic profiles of venoms were typically quite distinct from saliva profiles. Components from A. stolata, Hydrodynastes gigas, Tantilla nigriceps and T. e. vagrans venoms showed protease activity when run on gelatin zymogram gels. N-terminal protein sequences for three 26 kD venom components of three species (H. gigas, H. torquata, T. biscutatus) and one 3.5 kD component (T. nigriceps) were also obtained, and the 3.5 kD peptide showed apparent sequence homology with human vascular endothelial growth factor; these data represent the first sequences of colubrid venom components. Protease, phosphodiesterase and PLA₂ activities are also common to elapid and viperid snake venoms, but it is apparent that numerous other (as yet undescribed) components make up the majority of colubrid venom proteins. The complex nature of venoms produced by most species surveyed, and the high levels of protease or phospholipase A₂ activity of some venoms, suggest that many colubrids could become an important source of human health concern as encounters with these snakes increase.     

ELISA for the detection of toxic antigens in experimental and clinical envenoming by Tityus serrulatus scorpion venom

An ELISA was developed for identification of circulating toxic antigens from Tityus serrulatus scorpion venom. The toxic fraction from the scorpion venom was purified by Sephadex G-50 chromatography and immunoaffinity techniques were used for identifying antibodies that reacted with this fraction. These antibodies were used to develop a sandwich-type ELISA. The specificity of the assay was demonstrated by its capacity for identifying mice that were experimentally inoculated with T. serrulatus venom from those inoculated with Phoneutria nigriventer spider venom, Apis mellifera bee venom and Bothrops atrox, Crotalus durissus terrificus, Lachesis muta muta and Micrurus frontalis snake venoms. Measurable absorbance signals were obtained with 0.1 ng of venom per assay. The ELISA also detected antigens in the sera of patients systemically envenomed by T. serrulatus. Therfore, this ELISA could be a valuable tool for clinicians and epidemiologists, owing to its sensitivity and specificity.     

The renal effects of Bothrops jararacussu venom and the role of PLA2 and PAF blockers

The most common complication in the lethal cases of ophidian bites in Brazil is acute renal failure, but its pathogenesis is obscure. The effects of Bothrops jararacussu venom (3, 10 and 30 μg/ml) were examined using the isolated perfused kidney from Wistar rats. Dexamethasone, and WEB 2086, a triazolobenzodiazepine substance, which is a platelet activating factor receptor antagonist, were tested for a possible blockade of the renal effects in the presence of 10 μg/ml of venom. The most intense effects of the venom were noticed at 120 min after using 30 μg/ml. We observed a decrease in the perfusion pressure and in the renal vascular resistance. However, the glomerular filtration rate (GFR) and the urinary flow (UF) increased significantly. The percent of sodium (%Na_tot⁺) and potassium (%K_tot⁺) tubular transport were also decreased. Dexamethasone was unable to block the effects of B. jararacussu in the kidney, while WEB 2086 blocked its effect in glomerular filtration rate, urinary flow and in the percentage of total tubular potassium reabsorption. We suggest that this venom promotes diuresis independently of perfusion pressure drop. The alterations in GFR, UF and %K_tot⁺ are probably mediated by platelet activating factor. Dexamethasone did not block the renal effects maybe because of the concentration used in this work or maybe the renal effects are promoted by the myotoxin, which does not have PLA₂ activity.     

Citrate inhibition of snake venom proteases

Thirty snake venoms had a citrate content of 2.3 to 12.9%, dry basis, by an aconitase–isocitric dehydrogenase coupled enzyme assay. This is a venom concentration range of approximately 30 to 150 mM citrate assuming 25% venom solids content. Inhibition of snake venom protease activity by the addition of exogenous citrate was obtained using azure blue hide powder and azocasein as substrates. Protease inhibitions of 7.5% for Crotalus atrox venom to 78% for Bothrops picadoi venom were observed with citrate. Complete inhibition of snake venom protease activity by citrate was not observed. Bothrops asper (Pacifico) venom showed a 41% protease inhibition by citrate with azocasein as the substrate and 46% inhibition of Bothrops asper (Alantico) venom protease with azure blue hide power as a substrate. Trypsin was not inhibited in this system. Citrate may inhibit some venom protease activity by forming a complex with the zinc of zinc-dependent enzymes.     

Expression and characterization of a novel plasminogen activator from Agkistrodon halys venom

A venom gland cDNA library of Agkistrodon halys was constructed and screened with a probe based on the consensus sequence of venomic serine proteases. Next, we determined the sequences of the entire open reading frames of two selected positives which were found to encode novel serine proteases of 234 and 233 amino acids in length and named as Haly-PA and Haly 2, respectively. Upon protein data base search, Haly-PA showed the highest similarity of 82% to the previously characterized plasminogen activator, TSV-PA (Zhang et al. 1995, J. Biol. Chem., 270, 10246–10255). Haly 2 displayed a 78% similarity to β-fibrinogenase (Hung et al. 1994, B. B. R. C., 205, 1707–1715). Haly-PA was successfully expressed using the baculovirus system and secreted into the culture media as a 32 kDa glycoprotein. In the western analysis of snake venom, anti-Haly-PA antibody detected the same size of band indicating that this enzyme is a component of snake venom. Recombinant Haly-PA was purified to homogeneity using the combination of anion exchange and gel filtration column. In the fibrino(geno)lytic assay, recombinant Haly-PA displayed an indirect fibrino(geno)lytic activity depending on the presence of plasminogen and cleaved the plasminogen to generate the active plasmin. These results indicate that Haly-PA is a plasminogen activator and displays fibrino(geno)lytic activity through conversion of plasminogen to plasmin.     

Toona sinensis extracts induces apoptosis via reactive oxygen species in human premyelocytic leukemia cells

Toona sinensis (T. sinensis), well known in Taiwan as a traditional Chinese medicine, has been shown to exhibit antioxidant effects. In this study, therefore, the ability of T. sinensis to induce apoptosis was studied in cultured human premyelocytic leukemia HL-60 cells. Treatment of the HL-60 cells with a variety of concentrations of the aqueous extracts of T. sinensis (TS extracts) (10–75 μg/ml) and gallic acid (5–10 μg/ml), the natural phenolic components purified from TS extracts, resulted in dose- and time-dependent sequences of events marked by apoptosis, as shown by loss of cell viability and internucleosomal DNA fragmentation. Furthermore, apoptosis in the HL-60 cells was accompanied by the release of cytochrome c, caspase 3 activation and specific proteolytic cleavage of poly (ADP-ribose) polymerase (PARP). This increase in TS extracts- and gallic acid-induced apoptosis was also associated with a reduction in the levels of Bcl-2, a potent cell-death inhibitor, and an increase in those of the Bax protein, which heterodimerizes with and thereby inhibits Bcl-2. Interestingly, TS extracts- and gallic acid-induced dose-dependent reactive oxygen species (ROS) generation in HL-60 cells. We found that catalase significantly decreased TS extracts- or gallic acid-induced cytotoxicity, DNA fragmentation, and ROS production, however, slight reduction was observed with vitamins C and E. Our results indicate that TS extracts- or gallic acid-induced HL-60 apoptotic cell death could be due to the generation of ROS, especially H₂O₂. The data suggest that T. sinensis exerts antiproliferative action and growth inhibition on HL-60 cells through apoptosis induction, and, therefore, that it may have anticancer properties valuable for application in food and drug products.     

Isolation and characterisation of acanmyotoxin-2 and acanmyotoxin-3, myotoxins from the venom of the death adder Acanthophis sp. Seram

Death adder (genus Acanthophis) venoms display neurotoxic activity but were thought to be devoid of myotoxic components. Studies from our laboratory have shown that some species (i.e. Acanthophis rugosus and Acanthophis sp. Seram) posses venom with myotoxic activity [Wickramaratna JC, Fry BG, Aguilar M, Kini RM, Hodgson WC. Isolation and pharmacological characterisation of a phospholipase A₂ myotoxin from the venom of the Irian Jayan death adder (A. rugosus). Br J Pharmacol 2003;138:333–342; Wickramaratna JC, Fry BG, Hodgson WC. Species-dependent variations in the in vitro myotoxicity of death adder (Acanthophis) venoms. Toxicol Sci 2003;74:352–360]. The present study describes the isolation and characterisation of two myotoxins (acanmyotoxin-2 and acanmyotoxin-3) from A. sp. Seram venom. Venom was fractionated into approximately 12 major peaks using reverse phase high performance liquid chromatography. Two components caused concentration (0.1–1 μM) dependent inhibition of direct (2 ms, 0.1 Hz, supramaximal V) twitches and an increase in baseline tension in the chick biventer cervicis nerve-muscle. Histological examination of the muscle confirmed damage. PLA₂ activity was detected in both acanmyotoxin-2 (390.2 ± 19.7 μmol/(min mg); n = 4) and acanmyotoxin-3 (14.2 ± 7.7 μmol/(min mg); n = 4). In comparison, A. sp. Seram whole venom had a specific activity of 461.3 ± 90.4 μmol/(min mg) (n = 3). Mass spectrometry analysis indicated acanmyotoxin-2 had a mass of 13,082 Da and acanmyotoxin-2 13,896 Da. Acanmyotoxin-2 and acanmyotoxin-3 accounted for approximately 7 and 4% of total venom composition, respectively. N-terminal sequencing of the first 30 amino acids of each toxin indicated they shared some sequence homology with known myotoxins. In conclusion, clinicians should be aware that symptoms of envenoming by some species of death adder may include signs of myotoxicity as well as neurotoxicity. Future studies will investigate the efficacy of the current antivenom treatment against the myotoxic components of A. sp. Seram venom.     

Effects of Tityus serrulatus crude venom on the GABAergic and dopaminergic systems of the rat brain

This study was designed to investigate the effect of T. serrulatus scorpion venom on dopamine (DA) and gamma amino butyric acid (GABA) concentrations in different regions of the brain. The ratio of homovanillic acid (HVA) to DA, and the glutamic acid decarboxylase (GAD) activity were determined following intravenous or intracerebral venom injections. The increase in the HVA/DA ratio in the striatum after i.v. or intrastriatal injection could indicate an increase in DA turnover. One hour after i.v. injection of the venom GAD activity was shown to be decreased in the striatum and hypothalamus. After 24 hr GAD activity increased in the striatum and decreased in the hypothalamus and brain stem. These results could indicate different effects of the venom on the GABA system in different areas of the brain. After intrastriatal injection of the scorpion venom, the animals showed stereotyped behavior and rotation activity. Following intrahippocampal injection, myoclonus and orofacial automatisms, which constitute pro-convulsive signals, were observed. These behavioral alterations could be, at least in part, related to the GABA and dopamine alterations caused by the venom, since stereotypy, circling behavior and convulsions are dependent on dopamine and/or GABA.     

Expression, purification and molecular modeling of recombinant fibrinogenase [IV], a metalloproteinase from Deinakistrodon acutus venom.

A novel metalloproteinase, recombinant fibrinogenase IV (rFIV(a)), was expressed and purified from Deinakistrodon acutus venom. It was a single chain protein with an apparent molecular weight 27 kDa and an isoeletric point of pH 7.1. RFIV(a) cleaved preferentially the Aalpha-chain and also cleaved Bbeta, gamma-chains of fibrinogen when the incubation time was prolonged. The proteolytic activity was inhibited by EDTA, l-cysteine, and DTT, indicating rFIV(a) was a metalloproteinase requiring disulfide bonds for its activity. It kept above 85% of the initial activity from pH 4.5-11, showed an equal maximum activity at the temperature range from 30 to 50 degrees C, and was inactivated by Zn2+, Cu2+ and Cd2+. Homology modeling of rFIV(a) showed that two highly conserved disulfide bonds (Cys159-Cys164 and Cys117-Cys197) was maintained from its structure, and it exhibited the characteristic conserved motif H142E143XXH146XXGXXH152, whose three histidine residues were involved in binding of the catalytically essential zinc ion. This work demonstrates the expression, purification and characterization of recombinant fibrinogenase IV, which belongs to class P-I metalloproteinase from D. acutus venom.     

Immediate active movement following Bitis arietans or Naja mossambica venom injection diminishes or prevents necrosis in mice

Tourniquet use, which localises necrotising snake venom to the bite site, is suspected to exacerbate local necrosis. In view of this, the effect of immediate active movement on the outcome of necrotising snake venom injection was investigated in mice. A minimal macroscopic necrotic dose of Bitis arietans venom (2.5 μg) was administered subcutaneously into the dorsum of the right hind feet of 40 mice of which 20 were previously anaesthetised. The 20 non-anaesthetised mice swam for 10 min. All mice were euthanased on day 4. A further 40 mice underwent an identical experiment using 20 μg Naja mossambica venom and were euthanased on day 6. The areas of resulting macroscopic necrosis were measured in the B. arietans venom injected mice, while the largest diameter of macroscopic necrosis was determined in the N. mossambica venom injected mice. Immediate active movement (swimming) significantly prevented or reduced the area of necrosis in comparison to non-ambulant mice.     

Thioridazine treatment prevents cardiopathy in Trypanosoma cruzi infected mice

Trypanosoma cruzi trypanothione reductase is an enzyme that has been identified as a potential target for chemotherapy. Thioridazine inhibits it and prevented cardiopathy in mice infected with T. cruzi Tulahuen strain. As not all T. cruzi strains respond to treatment in the same way, an isolate from a chronic patient (SGO Z12) was used; parasitaemias were studied along with, survival, serology, electrocardiography, histology and cardiac β receptor function. Parasitaemia in thioridazine (80 mg/(kg day) for 3 days) treated mice was less and lasted for a shorter period (P<0.01), there were reduced electrocardiographic and histological alterations and significantly improved survival (80% of non-treated died). Treated mice had lower receptor affinity and higher density as a compensatory mechanism, modifying the course of T. cruzi infection (SGO Z12 isolate) and preventing the consequent cardiopathy.     

Molecular cloning, expression and immunological properties of LiD1, a protein from the dermonecrotic family of Loxosceles intermedia spider venom.

The present report describes the identification and molecular characterization of LiD1, a protein expressed in the venom gland of the brown spider Loxosceles intermedia. LiD1 belongs to a family of proteins with dermonecrotic activity and members of this family have been found in spiders from the genus Loxosceles. The necrotic lesions caused by this group of proteins may lead to serious socio-economic problems such as surgical tissue reconstitution and even patient death. LiD1 was cloned using a cDNA library constructed from the venom gland of L. intermedia and antibodies against proteins with dermonecrotic activity isolated from the crude venom of this spider. The amino acid sequence deduced from the cDNA revealed a mature protein of approximately 31 kDa, with a pI of 7.37. The cDNA also revealed the existence of a signal peptide, a propeptide and also an untranslated 3′ region with 218 nucleotides. LiD1 was expressed as a protein fused with β-galactoside protein using the vector pBK-CMV, resulting in the recombinant protein recLiD1 with important immunological properties. recLiD1 was strongly recognised by anti-dermonecrotic antibodies and was also able to generate reactive antibodies against native dermonecrotic proteins isolated from the venom of L. intermedia.     

A hyaluronidase from Potamotrygon motoro (freshwater stingrays) venom: Isolation and characterization

Freshwater stingrays (Potamotrygon motoro) are known to cause human accidents through a sting located in its tail. In the State of Goiás, this accident happens especially during the fishing season of the Araguaia River. The P. motoro venom extracted from the sting presented hyaluronidase activity. The enzyme was purified by gel filtration on Sephacryl S-100 and ion-exchange chromatography on SP-Sepharose. A typical procedure provided 376.4-fold purification with a 2.94% yield. The molecular weight of the purified enzyme was 79 kDa as estimated by gel filtration on Sephacryl S-100. The K_m and V_max values for hyaluronidase, using hyaluronic acid as substrate, were 4.91 μg/ml and 2.02 U/min, respectively. The pH optimum for the enzyme was pH 4.2 and maximum activity was obtained at 40 °C. The hyaluronidase from P. motoro was shown to be heat instable, being stabilized by bovine albumin and DTT, and inhibited by Fe²⁺, Mn²⁺, Cu²⁺ and heparin.