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An algorithm for automatic clustering of database protein sequences from Bothrops jararacussu venomous gland, according to sequence similarities of their domains, is described. The program was written in C and Perl languages. This algorithm compares a domain with each ORF protein sequence in the database. Each nucleotide FASTA sequence generates six ORFs. As a result, the user has a list containing all sequences found in a specific domain and a display of the sequence, domain and number of hits. The algorithm lists only the sequences that present a minimum similarity of 30 hits and the best alignment. This limit was considered appropriate. The algorithm is available in the Internet (www.compbionet.org.br/cgi-domains/homesnake) and it can quickly and accurately organizes large database into classes.

Two basic myotoxic PLA(2)s, namely BnpTX-I and II, were isolated from Bothrops neuwiedi pauloensis snake venom through three chromatographic steps: ion-exchange chromatography on CM-Sepharose, gel filtration on Sephadex G-50 and reverse phase HPLC on a C18 column. Both PLA(2)s showed a M(r) around 14,000 for the monomer and 28,000 for the dimer (as estimated by SDS-PAGE), pI approximately 7.8 and approximately 121 amino acid residues cross-linked by seven disulfide bonds. The N-terminal sequences revealed significant homology with Asp49 basic myotoxic PLA(2)s from other snake venoms. The catalytic and anticoagulant activities of BnpTX-I were higher than those of BnpTX-II. Both were able to induce cytotoxicity in vitro, as well as, myotoxicity, edema and lethality in mice. BnpTX-I also induced neurotoxic effect on mouse neuromuscular preparations and bactericidal activity on Eschericia coli and Staphylococcus aureus. After chemical modification of BnpTX-I with BPB or incubation with EDTA or Mn(2+) ions, the catalytic activity was completely abolished, while the toxic and pharmacological activities were partially reduced. Interaction with heparin inhibited the cytotoxic and bactericidal effects. Anti-BthTX-I, anti-BthTX-II and anti-115-129-C terminal antibodies strongly recognize both BnpTX-I and II. It is shown that the neurotoxic effect induced by B. neuwiedi pauloensis venom is due to the presence of myotoxic PLA(2)s. The data also corroborate the hypothesis of a partial dissociation between toxic and enzymatic domains. In addition, BnpTX-I displays a heparin binding C-terminal region, which is probably responsible for the cytotoxic and bactericidal effects.

The isolation and biochemical/enzymatic characterization of an L-amino acid oxidase, Balt-LAAO-I, from Bothrops alternatus snake venom, is described. Balt-LAAO-I is an acidic glycoprotein, pI approximately 5.37, homodimeric, Mr approximately 123,000, whose N-terminal sequence is ADVRNPLE EFRETDYEVL. It displays a high specificity toward hydrophobic and basic amino acids, while deglycosylation does not alter its enzymatic activity. Balt-LAAO-I induces platelet aggregation and shows bactericidal activity against Escherichia coli and Staphylococcus aureus. In addition, this enzyme is slightly hemorrhagic and induces edema in the mouse paw. Balt-LAAO-I is a multifunctional enzyme with promising relevant biotechnological and medical applications.

In order to better understand the function of acidic phospholipases A2 (PLA2s) from snake venoms, expressed sequence tags (ESTs) that code for acidic PLA2s were isolated from a cDNA library prepared from the poly(A)+ RNA of venomous glands of Bothrops jararacussu. The complete nucleotide sequence (366 bp), named BOJU-III, encodes the BthA-I-PLA2 precursor, which includes a signal peptide and the mature protein with 16 and 122 amino acid residues, respectively. Multiple comparison of both the nucleotide and respective deduced amino acid sequence with EST and protein sequences from databases revealed that the full-length cDNA identified (BOJU III--AY145836) is related to an acidic PLA2 sharing similarity, within the range 55-81%, with acidic phospholipases from snake venoms. Moreover, phylogenetic analysis of amino acid sequences of acidic PLA2s from several pit viper genera showed close evolutionary relationships among acidic PLA2s from Bothrops, Crotalus, and Trimeresurus. The molecular modeling showed structural similarity with other dimeric class II PLA2s from snake venoms. The native protein BthA-I-PLA2, a nontoxic acidic PLA2 directly isolated from Bothrops jararacussu snake venom, was purified and submitted to various bioassays. BthA-I-PLA2 displayed high catalytic activity and induced Ca2+-dependent liposome disruption. Edema induced by this PLA2 was inhibited by indomethacin and dexamethasone, thus suggesting involvement of the cyclo-oxygenase pathway. BthA-I-PLA2 showed anticoagulant activity upon human plasma and inhibited phospholipid-dependent platelet aggregation induced by collagen or ADP. In addition, it displayed bactericidal activity against Escherichia coli and Staphylococcus aureus and antitumoral effect upon breast adrenocarcinoma as well as upon human leukemia T and Erlich ascitic tumor. Following chemical modification with p-bromophenacyl bromide, total loss of the enzymatic and pharmacological activities were observed. This is the first report on the isolation and identification of a cDNA encoding a complete acidic PLA2 from Bothrops venom, exhibiting bactericidal and antitumoral effects.

Phospholipases A2 (PLA2s) constitute major components of snake venoms and have been extensively investigated not only because they are very abundant in these venoms but mainly because they display a wide range of biological effects, including neurotoxic, myotoxic, cytotoxic, edema-inducing, artificial membrane disrupting, anti-coagulant, platelet aggregation inhibiting, hypotensive, bactericidal, anti-HIV, anti-tumoral, anti-malarial and anti-parasitic. Due to this functional diversity, these structurally similar proteins aroused the interest of many researchers as molecular models for study of structure-function relationships. One of the main experimental strategies used for the study of myotoxic PLA2s is the traditional chemical modification of specific amino acid residues (His, Met, Lys, Tyr, Trp and others) and examination of the consequent effects upon the enzymatic, toxic and pharmacological activities. This line of research has provided useful insights into the structural determinants of the action of these enzymes and, together with additional strategies, supports the concept of the presence of 'pharmacological sites' distinct from the catalytic site in snake venom myotoxic PLA2s.

Lipases are versatile enzymes regarding the range of reactions they catalyse and substrates on which they act. They are as well important as catalyst in organic synthesis. Their immobilization on appropriate supports confer them greater stability besides the possibility of operating in continuous reactors. In order to explore these abilities, the reactions involving hydrolysis of p-nitrophenyl acetate (PNPA) and transesterification of PNPA with n-butanol were chosen. Lipases from two different sources were assayed, namely: microbial (Candida rugosa, CRL, Sigma Type VII) and pancreatic (PPL, Sigma, Type II). Two immobilization methods were also used, namely: 1). adsorption, using as support the following silica derivatives (150-300 microm e 450micro): phenyl, epoxy, amino and without derivation, and 2). covalent binding, using glutaraldehyde as binding agent and silica amino as support. This later method led to better results. Hydrolytic activity was 6.1 U/g(support) for CRL and 0.97 U/g(support) for PPL, and of transesterification, 2,8 U/g(support) for CRL and 1,9 U/g(support) for PPL. Stability of the immobilized enzyme as a function of temperature was evaluated for CRL at 40 degrees C and 50 degrees C and for PPL at 32 degrees C and 40 degrees C. The assays were initially carried out batchwise, both for soluble and immobilized enzymes, aiming to the obtention of parameters for the continuous reactor. Lipases immobilized by covalent binding were used in the assays of operational stability in continuous reactors. For PPL in aqueous medium, at 32 degrees C, and CRL in organic medium at 40 degrees C, both operating continuously, no significant loss of activity was detected along the analysis period of 17 days. In the case of CRL in aqueous medium at 40 degrees C there was a loss of activity around 40% after 18 days. For PPL in organic medium at 40 degrees C the loss was 33% after 20 days. Comparing both sources with each other, very different results were obtained. Higher activity was found for CRL, both for hydrolysis and for transesterification reactions, with higher stability in organic medium. PPL showed lower activity as well as higher stability in aqueous medium. The immobilization method by covalent binding showed to be the most appropriate. Immobilized lipases are therefore relatively stable both in aqueous and organic medium.

Spectrophotometric profiles representing the unfolding induced by guanidine on Bothrops moojeni myotoxins-I (MjTX-I) and II (MjTX-II), Bothrops jararacussu bothropstoxin-I (BthTX-I) and Bothrops pirajai piratoxin-I (PrTX-I) were obtained and compared with those obtained with bovine ribonuclease A (RNAse) and trypsin. The molar (epsilon(1M)) and percent (epsilon(1%)) extinction coefficients were determined for the four myotoxins as well as for RNAse and trypsin as reference parameters. These coefficients were then used throughout this work. The changes in free energy (deltaGD(H)(2)(O)) corresponding to zero guanidine concentration and the guanidine concentrations (D(1/2)) able to convert 50% of the molecules from the native to the unfolded state were determined. The values of deltaGD (H)(2)(O) ranged from 4.42 (BthTX-I) to 8.02 (MjTX-I) kcal/mole, compared with 6.47 and 6.88 kcal/mole for trypsin and RNAse, respectively. The values for deltaGD(H)(2)(O) and D1/2 showed that BthTX-I is the least stable among the four myotoxins assayed, with a D1/2 close to that of RNAse, while MjTX-II is conformationally the most stable. Monitoring of the unfolding of RNAse and PrTX-I by a 0 to 6 M urea gradient PAGE revealed transitions from the native (N) to the unfolded (U) state with deltaG(N-U)of 0.22 and 0.41 kcal/mole, respectively. Sigmoidal curves showed well-defined two-stage transitions for both proteins.

Bothropstoxin-I (BthTX-I), a myotoxic Lys49 phospholipase A(2) (PLA(2)) homologue isolated from Bothrops jararacussu snake venom, causes a range of biological effects, including myonecrosis, mouse paw edema, irreversible neuromuscular blockade and lysis of cell cultures. Among eight divalent cations assayed, Mn(2+) was the most effective in reducing mouse paw edema induced by BthTX-I (25 microg). Preincubating BthTX-I with Mn(2+) (1.0mM) reduced mouse paw edema by 70% and myotoxicity by 60% in mice injected i.m. with 50 microg toxin. Mn(2+) (50 microl of a 1mM solution) administered within 1min at the site of toxin injection was still but less effective in antagonising BthTX-I-induced myotoxicity. Mn(2+) (1.0mM) completely prevented BthTX-I (1.4 microM)-induced neuromuscular blockade in the mouse phrenic-nerve diaphragm preparation. Mn(2+) (0.25mM) protected about 85% of NB41A3 cells from lysis when coincubated with BthTX-I (1.0 microM) for 25h. Preincubation with 0.25mM Mn(2+) increased the sensitivity of the cells to subsequent lysis by BthTX-I in the absence of Mn(2+). BthTX-I (1 microM) caused extensive fatty acid release (from 0.8% of the total radiolabeled lipid in control cells to 56% with toxin) when incubated with the NB41A3 cell line for 25h. PLA(2) activity observed in cell cultures after addition of BthTX-I was considerably reduced by 0.25mM Mn(2+). Mn(2+) ions constitute a promising agent to assess the action mechanism and the effects of enzymatic inhibition on the pharmacological activity of Lys49 PLA(2) homologues.