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Made available in DSpace on 2014-12-03T13:11:08Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-03-01Bitstream added on 2014-12-03T13:22:45Z : No. of bitstreams: 1 WOS000335755700005.pdf: 1237419 bytes, checksum: 63efa3a070a9fc1de02aeccb01a674cd (MD5) Deutsche Forschungsgemeinschaft Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Higher Education Commission of Pakistan German Academic Exchange Service (DAAD), Germany Alexander von Humboldt Foundation Elapid snake venom is a highly valuable, but till now mainly unexplored, source of pharmacologically important peptides. We analyzed the peptide fractions with molecular masses up to 10 kDa of two elapid snake venoms-that of the African cobra, N. m. mossambica (genus Naja), and the Peninsula tiger snake, N. scutatus, from Kangaroo Island (genus Notechis). A combination of chromatographic methods was used to isolate the peptides, which were characterized by combining complimentary mass spectrometric techniques. Comparative analysis of the peptide compositions of two venoms showed specificity at the genus level. Three-finger (3-F) cytotoxins, bradykinin-potentiating peptides (BPPs) and a bradykinin inhibitor were isolated from the Naja venom. 3-F neurotoxins, Kunitz/basic pancreatic trypsin inhibitor (BPTI)-type inhibitors and a natriuretic peptide were identified in the N. venom. The inhibiting activity of the peptides was confirmed in vitro with a selected array of proteases. Cytotoxin 1 (P01467) from the Naja venom might be involved in the disturbance of cellular processes by inhibiting the cell 20S-proteasome. A high degree of similarity between BPPs from elapid and viperid snake venoms was observed, suggesting that these molecules play a key role in snake venoms and also indicating that these peptides were recruited into the snake venom prior to the evolutionary divergence of the snakes. Univ Hamburg, Inst Biochem & Mol Biol, Lab Struct Biol Infect & Inflammat, DESY, D-22603 Hamburg, Germany Univ Engn & Technol, Dept Chem, Lahore 54890, Pakistan Univ Hamburg, Inst Organ Chem, D-20146 Hamburg, Germany Univ Med Ctr Hamburg Eppendorf UKE, Inst Clin Chem, D-20246 Hamburg, Germany IBILCE UNESP, Dept Phys, BR-15054000 Sao Jose Do Rio Preto, SP, Brazil Inst Pesquisas Energet & Nucl, Ctr Biotecnol, BR-05508000 Sao Paulo, Brazil IBILCE UNESP, Dept Phys, BR-15054000 Sao Jose Do Rio Preto, SP, Brazil Deutsche ForschungsgemeinschaftBE 1443-18-1 Deutsche Forschungsgemeinschaft26-1 Alexander von Humboldt Foundation3.3-BUL/1073481 STP

We report the development of molecularly imprinted polyacrylamide nanoparticles that bind to and neutralize the activity of cytotoxins present in the venom of the Mozambique Spitting Cobra (Naja mossambica mossambica). The binding activity of these nanoparticles is avid and specific. These findings hold promise for the development of a synthetic antivenom.

It has been found that the lethal action of elapid snake venoms to arthropods (fly larvae and isopods) is due to proteic factors differing from the toxins which are strongly and specifically active on mammals. This conclusion was based on the following: (1) Lack of any correlation between the toxic activity on larvae, isopods, and mice of ten elapid snake venoms. (2) Absence of any toxicity to arthropods in pure toxins isolated and purified from several elapid snake venoms according to their lethality. (3) Electrophoretical separation of the venom of the snake Naja mossambica mossambica (= N. nigricollis mossambica) resulted in fractions active either to arthropods and/or to mice. (4) Separation of the above venom by gel filtration on Sephadex G-50 enabled the isolation of fractions highly toxic to arthropods. (5) The above fractions demonstrated a high phospholipase activity corresponding to about 80 per cent of the total activity of the whole venom. The link between phospholipase and toxicity to arthropods will serve as a target for further investigation. It appears that the phenomenon of diversity in toxic activities of different proteins to different groups of organism, as previously demonstrated in scorpion venoms, is equally shared by elapid snake venoms.

Summary The primary structure of cytotoxin VII4, consisting of 60 amino acid residues cross-linked by four disulphide bridges, was determined. Cytotoxin VII4 differs in altogether 17 residue positions from the other cytotoxins of the same snake. Comparing the amino acid sequence of cytotoxin VII4 from Naja mossambica mossambica venom to the known sequences of cytotoxins from the genera Naja and Haemachatus , 33 residues, including 8 half cystines, are identical. The amino acid sequence of cytotoxins as a group has 11 residue positions in common with the amino acid sequence of neurotoxins and based on this observation, 8 residues in the primary structure of cytotoxins could be implicated as probably being functionally important.

Three phospholipases A, CM-I, CM-II and CM-III, were purified from Naja mossambica mossambica venom by gel filtration on Sephadex G-50 followed by ion-exchange chromatography on CM-cellulose. They comprise each 118 amino acid residues and are cross-linked by seven intrachain disulphide bridges. The complete primary structure of the three phospholipases A have been elucidated. The sequences and the invariant amino acid residues of CM-I, CM-II and CM-III resemble those of phospholipases A from other snake venoms and also from porcine pancreas. However, the letality (LD50 values) of the three phospholipases A from Naja mossambica mossambica venom, differ among themselves, and are also much higher than the LD100 value encountered for notexin from Notechis scutatus scutatus venom.

The monomolecular film technique was used in order to study the specific interactions of 4 cardiotoxins from Elapidae snake venom Naja mossambica mossambica with different phospholipids. The interaction, at pH 7.5, of cardiotoxin (10-7M) with both neutral and negatively charged phospholipids occurs up to a very high critical surface pressure ( π = 45 dynes/cm with the latest). The apparent molecular area of cardiotoxin molecule, during its insertion into a negatively charged phospholipid film, presents only two characteristic values : 1400 A2 for π dynes/cm and 420 A2 for π > 30 dynes/cm , the transition occuring in a very narow range of surface pressure ( 25 ± 5 dynes/cm ). Thus, according to the surface pressure, the cardiotoxin may present two different orientations : “flat” or “edgewise” and the transition between both could account for lytic activity of cardiotoxin.

The monomolecular film technique was used to compare the specific interaction of four cardiotoxins from Naja mossambica mossambica with different phospholipids. We were able to demonstrate the interaction of cardiotoxins (10(-7) M) with both neutral and negatively charged phospholipids up to very high surface pressures (45 dyn/cm). In the presence of a phospholipid monolayer, the surface activity of cardiotoxins became much greater than that observed at the air-water interface. Neurotoxins of the same venom do not penetrate a phospholipid film, even at low surface pressure (15 dyn/cm). The apparent molecular area of cardiotoxin III during its insertion into a negatively charged phospholipid film was quantitatively defined. As a function of surface pressure of the membrane around 25 dyn/cm, cardiotoxins may exist in two different configurations, "flat" (1400 A2) or "edgewise" (420 A2). This result could account for the lytic activity of this type of toxin.

Five non-neurotoxic polypeptides, designated VII1–VII4 and V 5, have been isolated from the venom of Naja mossambica mossambica by column chromatographic techniques. Whole venom was fractionated on Sephadex G-50 and the major fraction, thus obtained, was re-chromatographed on CM-cellulose and Amberlite CG-50 to yield the polypeptides in pure form. They consist of 60 amino acid residues each and have molecular weights of 6820, 6792, 6888, 6712 and 6991, respectively. Immunochemically, polypeptides VII1–VII4 could be identified as cardiotoxins. The position with respect to Polypeptide V 5 was unclear and did not allow a definite assignment. This is the first time that the isolation of more than two cardiotoxins from the venom of the same specie has been described. Polypeptide VII1 was found to have the same amino acid composition as toxin γ, a cardiotoxin that has been isolated from the venom of Naja mossambica pallida.

International audience; High-resolution phase-sensitive two-dimensional proton nuclear magnetic resonance was used to monitor the preparation by high-performance liquid chromatography of homogeneous proteins from the venom of Naja mossambica mossambica. This resulted in the characterization of a heterogeneous protein preparation VII2, which had been used in earlier structural studies by NMR, as well as a homogeneous protein CTXIIb and a nearly homogeneous protein fraction CTXIIa, which are now both subject to further investigations of their solution conformations.

Proton n.m.r. spectra at 360 MHz of neurotoxin II and cardiotoxin VII4 from the venom of Naja mossambica mossambica are reported. From the n.m.r. spectra the solution conformations of the two proteins seem to be quite closely related. However, the exchange rates of the n.m.r. observable labile protons with deuterium of the solvent were markedly different, showing that the molecular structure of the cardiotoxin must be more flexible than that of the neurotoxin and suggesting that the different functional properties of the two toxins might be related to the different molecular dynamics.