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Snake venoms are natural sources of biologically active molecules that are able to act selectively and specifically on different cellular targets, modulating physiological functions. Thus, these mixtures, composed mainly of proteins and peptides, provide ample and challenging opportunities and a diversified molecular architecture to design and develop tools and agents of scientific and therapeutic interest. Among these components, peptides and small proteins play diverse roles in numerous physiological processes, exerting a wide range of pharmacological activities, such as antimicrobial, antihypertensive, analgesic, antitumor, analgesic, among others. The pharmaceutical and cosmetic industries have recognized the huge potential of these privileged frameworks and believe them to be a promising alternative to contemporary drugs. A number of natural or synthetic peptides from snake venoms have already found preclinical or clinical applications for the treatment of pain, hypertension, cardiovascular diseases and aging skin. A well-known example is captopril, whose natural peptide precursor was isolated from Bothrops jararaca snake venom, which is a peptide-based drug that inhibits the angiotensin-converting enzyme, producing an anti-hypertensive effect. The present review looks at the main peptides (natriuretic peptides, bradykinin-potentiating peptides and sarafotoxins) and low mass proteins (crotamine, disintegrins and three-Finger toxins) from snake venoms, as well as synthetic peptides inspired by them, describing their biochemical, structural and physiological features, as well as their applications as research tools and therapeutic agents.

DrTI was effective against trypsin-like enzymes from A. kuehniella and C. cephalonica, however an artificial diet was insufficient to affect the survival and body weight of either insect. The inhibitor stimulated chymotrypsin-like enzymes and probably induced the synthesis of enzymes insensitive to TLCK in neonate larvae.

Cr 5 PLA2 homologous (K49) was isolated from Calloselasma rhodostoma venom in only one chromatographic step in reverse phase HPLC (RP-HPLC) (on μ-Bondapack C-18). A molecular mass of 13.965 Da was determined by MALDI-TOF mass spectrometry. The amino acid composition showed that Cr 5 had a high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical residues of a basic PLA2. The complete amino acid sequence of Cr 5 PLA2 contains 120 residues, resulting in a calculated pI value of 5.55. This sequence shows high identity values when compared to other K49 PLA2s isolated from the venoms of viperid snakes. Lower identity is observed in comparison to D49 PLA2s. The sequence found was SLVELGKMIL QETGKNPAKS YGAYGCNCGV LGRHKPKDAT DRCCFVHKCC YKKLTGCDPK KDRYSYSWKD KTIVCGENNP CLKEMCECDK AVAICLRENL DTYNKKYRYL KPFCKKADDC. In mice, Cr 5 induced myonecrosis and edema upon intramuscular and intravenous injections, respectively. The LD50 of Cr 5 was 0.070 mg/kg of the animal weight, by intracerebroventricular (i.c.v.) route. In vitro, the toxin caused rapid cytolytic effect upon mouse skeletal muscle myoblasts in culture. The isolation of this PLA2 and the combined structural and functional information obtained classify Cr 5 as a new member of the K49 PLA2 family, since it presents typical features from such proteins.

In this article we investigated the platelet aggregating activity of whole crotoxin and its subunits isolated from Crotalus durissus cascavellavenom. During the purification protocols of the venom, using HPLC molecular exclusion, we detected the presence of two different serine protease activities in the gyroxin fraction, and another in the crotoxin fraction, which induced strong and irreversible platelet aggregation, in addition to blood coagulation. From crotoxin, we isolated PLA2, crotapotin (both fractions corresponding approximately 85% of whole crotoxin) and another minor fraction (F20) that exhibited serine protease activity. After a new fractionation on reverse phase HPLC chromatography, we obtained three other fractions named as F201, F202 and F203. F202 was obtained with high degree of molecular homogeneity with molecular mass of approximately 28 kDa and a high content of acidic amino residues, such as aspartic acid and glutamic acid. Other important amino acids were histidine, cysteine and lysine. This protein exhibited a high specificity for BApNA, a Michaelis-Menten behavior with Vmax estimated in $$5.64\,\upmu$$M/min and a Km value of 0.58 mMfor this substrate. In this work, we investigated the ability of F202 to degrade fibrinogen and observed α and β chain cleavage. Enzymatic as well as the platelet aggregation activities were strongly inhibited when incubated with TLCK and PMSF, specific inhibitors of serine protease. Also, F202 induced platelet aggregation in washed and platelet-rich plasma, and in both cases, TLCK inhibited its activity. The N-terminal amino acid sequence of F202 presented a high amino acid sequence homology with other thrombin-like proteins, but it was significantly different from gyroxin. These results showed that crotoxin is a highly heterogeneous protein composed of PLA2, thrombin-like and other fractions that might explain the diversity of physiological and pharmacological activities of this protein.In this article we investigated the platelet aggregating activity of whole crotoxin and its subunits isolated from Crotalus durissus cascavellavenom. During the purification protocols of the venom, using HPLC molecular exclusion, we detected the presence of two different serine protease activities in the gyroxin fraction, and another in the crotoxin fraction, which induced strong and irreversible platelet aggregation, in addition to blood coagulation. From crotoxin, we isolated PLA2, crotapotin (both fractions corresponding approximately 85% of whole crotoxin) and another minor fraction (F20) that exhibited serine protease activity. After a new fractionation on reverse phase HPLC chromatography, we obtained three other fractions named as F201, F202 and F203. F202 was obtained with high degree of molecular homogeneity with molecular mass of approximately 28 kDa and a high content of acidic amino residues, such as aspartic acid and glutamic acid. Other important amino acids were histidine, cysteine and lysine. This protein exhibited a high specificity for BApNA, a Michaelis-Menten behavior with Vmax estimated in $$5.64\,\upmu$$M/min and a Km value of 0.58 mMfor this substrate. In this work, we investigated the ability of F202 to degrade fibrinogen and observed α and β chain cleavage. Enzymatic as well as the platelet aggregation activities were strongly inhibited when incubated with TLCK and PMSF, specific inhibitors of serine protease. Also, F202 induced platelet aggregation in washed and platelet-rich plasma, and in both cases, TLCK inhibited its activity. The N-terminal amino acid sequence of F202 presented a high amino acid sequence homology with other thrombin-like proteins, but it was significantly different from gyroxin. These results showed that crotoxin is a highly heterogeneous protein composed of PLA2, thrombin-like and other fractions that might explain the diversity of physiological and pharmacological activities of this protein.

In this paper we reported the purification, the biological characterization and the amino acid sequence of two new isoforms basic 6-1 (Bj-IV) and 6-2 (Bj-V) PLA2D49 purified from the Bothrops jararacussuvenomIn this paper we reported the purification, the biological characterization and the amino acid sequence of two new isoforms basic 6-1 (Bj-IV) and 6-2 (Bj-V) PLA2D49 purified from the Bothrops jararacussuvenom

A lectin from Delonix regia(DRL) seeds was purified by gel filtration on Sephadex G-100 followed by ion-exchange chromatography on diethylaminoethyl-Sepharose and reverse-phase high-performance liquid chromatography on a C18 column. Hemagglutinating activity was monitored using rat erythrocytes. DRL showed no specificity for human erythrocytes of ABO blood groups. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a single protein in the presence of 0.1 M of dithiothreitol (DTT) and in nonreducing conditions. Native-PAGE showed that DRL is a monomer with a molecular mass of about 12 kDa, as determined by denaturing gel electrophoresis and gel filtration chromatography. An amino acid composition revealed the absence of cysteine residues, the presence of 1 mol methionine/mol protein and a high proportion of acidic amino acids and glycine. The N-terminal sequence of DRL was determined by Edman degradation, and up to 16 amino acid residues showed more than 90% homology with other lectins from the Leguminosae family. The optimal pH range for lectin activity was between pH 8.0 and 9.0, and the lectin was active up to 60°C. The lectin required Mn2+for hemagglutinating activity and remained active after reduction with 0.1 M of DTT, but lost activity in the presence of 8 M of urea. Sodium metaperiodate had no effect on the activity of DRL.

The venom of Crotalus durissus terrificuswas fractionated by reverse-phase HPLC to obtain crotapotins (F5 and F7) and PLA2(F15, F16, and F17) of high purity. The phospholipases A2(PLA2s) and crotapotins showed antimicrobial activity against Xanthomonas axonopodis pv. passiflorae,although the unseparated crotoxin did not. The F17 of the PLA2also revealed significant anticoagulant activity, althrough for this to occur the presence of Glu 53 and Trp 61 is important. The F17 of the PLA2showed allosteric behavior in the presence of a synthetic substrate. The amino acid sequence of this PLA2isoform, determined by automatic sequencing, was HLLQFNKMLKFETRKNAVPFYAFGCYCGWGGQRRPKDATDRCCFVHDCCYEKVTKCNTKWDFYRYSLKSGYITCGKGTWCKEQICECDRVAAECLRRSLSTYKNEYMFYPDSRCREPSETC. Analysis showed that the sequence of this PLA2isoform differed slightly from the amino acid sequence of the basic crotoxin subunit reported in the literature. The homology with other crotalid PLA2cited in the literature varied from 60% to 90%. The pL was estimated to be 8.15, and the calculated molecular weight was 14664.14 as determined by Tricine SDS-PAGE, two-dimensional electrophoresis, and MALDI-TOFF. These results also suggested that the enzymatic activity plays an important role in the bactericidal effect of the F17 PLA2as well as that of anticoagulation, although other regions of the molecule may also be involved in this biological activity.

A crotoxin homolog was purified from the Crotalus durissus collilineatusvenom using molecular exclusion and reverse-phase HPLC. This crotoxin contained one PLA2(Cdcolli III F6) and four crotapotin isoforms, whereas crotoxin from Crotalus durissus terrificusvenom had three PLA2isoforms and two crotapotin isoforms. SDS-PAGE showed that the C. d. collilineatusPLA2and crotapotin had relative molecular mass of 15 and 9 kDa, respectively. Neither the PLA2(Cdcolli III F6) nor the crotapotins (Cdcolli III F3 and F4) had any neurotoxicity in mouse phrenic nerve-diaphragm preparations when tested alone. However, when PLA2and crotapotin were coincubated before testing, the neurotoxicity was restored to a level similar to test in the venom in native crotoxin. The two crotapotins (Cdcolli III F3 and F4) differed in their ability to inhibit PLA2activity, perhaps because of variations in their affinities for this enzyme. Cdcolli III F6 showed allosteric enzymatic behavior, with maximal activity at pH 8.3 and 36°C. Full PLA2activity required the presence of a low Ca2+concentration and was inhibited by Cu2+and Zn2+and by Cu2+and Mg2+in the presence and absence of Ca2+, respectively. These results indicate that crotoxin from C. d. collineatusvenom is very similar enzymatically to crotoxin from C. d. terrificus.

A novel basic phospholipase A2(PLA2) isoform was isolated from Bothrops jararacussusnake venom and partially characterized. The venom was fractionated by HPLC ion-exchange chromatography in ammonium bicarbonate buffer, followed by reverse-phase HPLC to yield the protein Bj IV. Tricine SDS-PAGE in the presence or absence of dithiothreitol showed that Bj IV had a molecular mass of 15 and 30 kDa, respectively. This enzyme was able to form multimeric complexes (30, 45, and 60 kDa). Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The N-terminal sequence (DLWSWGQMIQETGLLPSYTTY . . .) showed a high degree of homology with basic D49 PLA2myotoxins from other Bothropsvenoms. Bj IV had high PLA2activity and produced moderate myonecrosis in skeletal muscle, but showed no neuromuscular activity in mouse phrenic nerve-diaphragm preparations. Bj IV showed allosteric enzymatic behavior, with maximal activity at pH 8.2 and 35-45°C. Full PLA2activity required Ca2+but was inhibited by Cu2+and Zn2+, and by Cu2+and Mg2+in the presence and absence of Ca2+, respectively. Crotapotins from Crotalus durissus terrificusrattlesnake venom significantly inhibited the enzymatic activity of Bj IV. The latter observation suggested that the binding site for crotapotin in this PLA2was similar to that in the basic PLA2of the crotoxin complex from C. d. terrificusvenom. The presence of crotapotin-like proteins capable of inhibiting the catalytic activity of D49 PLA2could partly explain the low PLA2activity of Bothropsvenoms.

Two new trypsin inhibitors, TDI-I and TDI-II, were purified from the seeds of the native Brazilian tree Copaifera langsdorffii (Caesalpinoideae, Leguminosae). The purification procedure involved ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Sepharose, affinity chromatography on trypsin-Sepharose, and reversed-phase (RP) HPLC. RP-HPLC yielded two forms (TDI-I and TDI-II), as confirmed by isoelectric focusing, with pI values between 7.0 and 8.1. The molecular mass of the TDI forms was 24 kDa based on FPLC gel filtration on Superdex 75. Under reducing conditions in tricine SDS-PAGE the molecular masses of TDI-I and TDI-II were 12 and 10 kDa, respectively. The Kivalues were 1.1 and 1.2 nM for TDI-I and TDI-II, respectively, and there was no inhibitory effect on chymotrypsin. Amino acid analysis revealed high levels of aspartic acid, glutamic acid, serine, glycine, proline, and lysine but low levels of methionine and aromatic amino acids in both inhibitors; the calculated molecular masses were 11,456 and 10,008 for TDI-I and II, respectively. Based on the N-terminal sequences of TDI-I and TDI-II, TDI-I belongs to the Kunitz family of trypsin inhibitors, whereas TDI-II showed no homology to any other protein. This observation suggests that TDI-II belongs to a new inhibitor subclass of low-molecular mass proteins in the subfamily Caesalpinoideae.

The PLA2and crotapotin subunits of crotoxin from Crotalus durissus cascavellavenom were purified by a combination of high-performance liquid chromatography (HPLC) molecular exclusion (Protein Pack 300SW column) and reverse-phase HPLC (RP-HPLC). Tricine SDS-PAGE showed that the PLA2and crotapotins migrated as single bands with estimated molecular masses of 15 and 9 kDa, respectively. The amino acid composition of the PLA2showed the presence of 14 half-cysteines and a high content of basic residues (Lys, Arg, His), whereas the crotapotins were rich in hydrophobic, negatively charged residues and half-cysteines. The PLA2showed allosteric behavior, with maximal activity at pH 8.3 and 35–40°C. C. d. cascavellaPLA2required Ca2+for activity but was inhibited by Cu2+and Zn2+and by Cu2+and Mg2+in the presence and absence of Ca2+, respectively. Crotapotin (F3) and heparin inhibited the catalytic activity of the PLA2by acting as allosteric inhibitors.

The PLA2and crotapotin subunits of crotoxin from Crotalus durissus cascavellavenom were purified by a combination of HPLC molecular exclusion (Protein Pack 300SW column) and reverse-phase HPLC (RP-HPLC). Tricine SDS—PAGE showed that the PLA2and crotapotins migrated as single bands with estimated molecular masses of 15 and 9 kDa, respectively. The amino acid composition of the PLA2showed the presence of 14 half-cysteines and a high content of basic residues (Lys, Arg, His), whereas the crotapotins were rich in hydrophobic, negatively charged residues and half-cysteines. The PLA2showed allosteric behavior, with maximal activity at pH 8.3 and 35–40°C. The C. d. cascavellaPLA2required Ca2+for activity, but was inhibited by Cu2+and Zn2+and by Cu2+and Mg2+in the presence and absence of Ca2+, respectively. Crotapotin (F3) and heparin inhibited the catalytic activity of the PLA2by acting as allosteric inhibitors.

1) The Soret region absorption spectrum of erythrocruorin (ERC) obtained from Glossoscolex paulistus, shows that oxy‐ERC has a maximum absorption peak at 416 nm while the deoxy‐ERC form has a maximum at 427 nm. 2) In the presence of a specific antiserum (anti‐ERC) and of anti‐ERC immunoglobulin G raised in rabbits, there is a deviation to low wavelengths in the maximum absorption peak of deoxy‐ERC while for the oxy form a red‐shift is noticed. These shifts accompanied an increased affinity of the hemeprotein for oxygen, possibly because of changes in the overall macromolecular conformation. 3) A decrease in the oxygen affinity of erythrocruorin is observed when large amounts of non‐specific serum are used. The same effect is observed in the presence of serum albumin, probably as a result of non‐specific binding between the albumin and erythrocruorin. 4) The fluorimetric titration of erythrocruorin with anti‐ERC Fab fragments results in a decrease in the intrinsic tryptophan fluorescence of the hemeprotein, a response indicative of a modification in the ERC's quaternary structure.

The complete amino acid sequence of bothropstoxin-II (BthTX-II), a myotoxin isolated from Bothrops jararacussusnake venom, is reported. The results show that BthTX-II is an Asp-49 phospholipase A2(PLA2)-like protein composed of a single polypeptide chain of 120 amino acid residues (Mr= 13,976), containing one methionine and 14 half-cystines. Despite a high degree of homology with other PLA2's and the presence of the strategic residues known to compose the Ca2+-binding loop, namely Tyr-28, Gly-30, Gly-32, and especially Asp-49, besides His-48, Tyr-52, and Asp-99, all of them directly or indirectly involved in catalysis, BthTX-II revealed a very low PLA2activity when assayed on egg yolk phosphatidylcholine. We attribute this low catalytic activity to the existence of extra mutations, e.g., Trp-5 for Phe-5, which points to the need of considering other strategic positions, since only Lys-49 PLA2's have been considered to be devoid of this enzymatic activity.

Bothrops jararacussu venom contains a new potent hemagglutinin (BJcuL), isolated by affinity chromatography on immobilized D‐galactose. Hemagglutination activity of BJcuL against pig erythrocytes was inhibited by galactosides, EDTA or EGTA. The purified lectin is a di sulfide dimer composed of 15 kDa subunits. Association of these subunits is essential for lectin activity, since DTT‐reduced BJcuL showed no hemagglutination activity against pig erythrocytes. Reduced and S‐pyridylethylated BJcuL (RP‐BJcuL) was separated as a single peak by reverse‐phase HPLC on a C‐18/μBondapak column. Results of the application of Edman degradation methodology showed that RP‐BJcuL has a single N‐terminal amino acid sequence, indicating that BJcuL is a homodimer. The N‐terminal 8‐residue (NNCPQDWL) shows homology with other lectins from snake venoms.

The complete amino acid sequence of bothropstoxin-I (BthTX-I), a myotoxin isolated fromBothrops jararacussusnake venom, is reported. The results show that BthTX-I is a Lys49phospholipase A2(PLA2)-like protein composed of a single polypeptide chain of 121 amino acid residues (Mr=13,720), containing one methionine and 14 half-cystines. Although deprived of any detectable PLA2activity, BthTX-I reveals a high degree of sequence homology with Asp49-PLA2sand with other Lys49-myotoxins. Critical mutations—such as Leu5for Phe5; Gln11for X11; Asn28for Tyr28; Leu32for Gly32; Lys49for Asp49; and Asp71for Asn71—which are apparently involved with the decreasing or elimination of PLA2activity, have been detected. The same mutations occurred in myotoxin II fromBothrops aspervenom, but five extra changes—namely, Pro90for Ser90; Gly111for Asn111; His120for Tyr120; Phe124for Leu124; and Pro132for Ala132—have been found relative to myotoxin II.

The Kunitz‐type trypsin inhibitor from seeds of Flamboyant (Delonix regia) has been purified to homogeneity and plate‐like crystals suitable for X‐ray analysis have been grown by the hanging‐drop method using PEG 6000 as a precipitant. The crystals belong to space group P212121with unit‐cell parameters a= 32.15, b= 69.39, c= 72.54 Å. X‐ray diffraction data have been collected to 2.95 Å resolution. The structure has been solved by molecular replacement using the known structures of trypsin inhibitors from Erythrina caffraseeds (PDB code 1tie) and from soya beans (Glycine max; PDB code 1ba7) as search models.

The phospholipases A2(PLA2, E.C. 3.1.1.4, phosphatide sn2 acylhydrolases) are the major components of the venom of several snakes. They are responsible for several important pharmacological effects observed in ophidian incidents. PLA2piratoxin II from Bothrops pirajaihas been crystallized by the vapour‐diffusion technique. X‐ray diffraction data have been collected to 2.04 Å resolution (90.2% complete, Rmerge= 0.070). The space group is P21and the cell parameters are a= 46.19, b= 60.36, c= 58.74 Å and β = 96.05°. The structure has been solved by molecular replacement using the crystallographic structure of PLA2from Bothrops asper(PDB code 1CLP) as a search model.

Scorpion neurotoxin Ts‐γ was isolated from Tityus serrulatusvenom and purified to apparent homogeneity by ion‐exchange HPLC. Crystals of the toxin were grown using polyethylene glycol 6000 as precipitant and were found to belong to the monoclinic space group P21with cell parameters a= 22.20, b= 36.90, c= 31.57 Å, β = 100.85°. The crystals diffract beyond 1.73 Å resolution at a synchrotron beamline, being notably stable during X‐ray exposure. The structure has been solved by molecular replacement using the very high resolution structure of Sahara scorpion Androctonus australisHector (PDB code 1AHO) as a search model.