Purification and amino acid sequences of six Tx3 type neurotoxins from the venom of the Brazilian 'armed' spider Phoneutria nigriventer (Keys).

Six neurotoxic peptides (Tx3-1 to Tx3-6) were purified from the venom of the spider Phoneutria nigriventer by a combination of gel filtration, reverse phase FPLC on PEP-RPC and PRO-RPC columns, reverse phase HPLC on Vydac C18, and ion exchange HPLC on cationic and anionic columns. These toxins caused different neurological symptoms in mice after intracerebroventricular injection. At dose levels of 5 micrograms/mouse, Tx3-3 and Tx3-4 caused rapid general flaccid paralysis followed by death in 10-30 min; Tx3-2 induced immediate clockwise gyration and flaccid paralysis after 6 hr; Tx3-1, Tx3-5 and Tx3-6 produced paralysis only in the posterior limbs and gradual decreases in movement and aggression during 24 hr. The mol. wt of these cystine-rich peptides were found to be in the range of 3500-8500 by mass spectroscopy and SDS-PAGE. The complete amino acid sequences of the neurotoxins Tx3-1 (40 residues), Tx3-2 (34 residues) and Tx3-6 (55 residues), and the N-terminal sequences of Tx3-3 (34 res.), Tx3-4 (40 res.) and Tx3-5 (36 res.) were established by direct automated Edman degradation, and manual DABITC/PITC microsequence analyses of peptides obtained from digests with various proteases. The structures of these Tx3 neurotoxins from Phoneutria nigriventer exhibited sequence similarities to one another and to the neurotoxins from the venoms of the spiders Hololena curta and Agelenopsis aperta, which were most evident in the location of the Cys residues.

The purification and amino acid sequences of four Tx2 neurotoxins from the venom of the Brazilian 'armed' spider Phoneutria nigriventer (Keys).

Four neurotoxic polypeptides (Tx2-1, Txt2-5, Tx2-6 and Tx2-9) were purified from the venom of the South American 'armed' spider Phoneutria nigriventer (Keys) by gel filtration and reverse phase FPLC and HPLC. These cysteine-rich polypeptides exhibited different levels of neurotoxicity in mice after intracerebroventricular injection. Tx2-1, Tx2-5 and Tx2-6 caused spastic paralysis and death, but the less toxic Tx2-9 produced only tail erection and scratching. The molecular weights of the polypeptides as determined by desorption mass spectroscoopy were 5838.8 for Tx2-1, 5116.6 (Tx2-5), 5291.3 (Tx2-6) and 3742.1 (Tx2-9). The complete amino acid sequences of the neurotoxins were determined by automated Edman degradation and by manual DABITC-PITC microsequence analysis of peptides obtained after digestions with various proteases. The amino acid sequences of Tx2-1 (53 residues), Tx2-5 (49 residues) and Tx2-6 (48 residues) were homologous, but had only limited similarities to the less toxic Tx2-9 (32 residues). All four polypeptides had varying sequence identities with other neurotoxins from different spider species and biologically active peptides from scorpions, a sea snail and seeds of Mirabilis jalapa.

The purification and amino acid sequence of the lethal neurotoxin Tx1 from the venom of the Brazilian 'armed' spider Phoneutria nigriventer.

A lethal neurotoxic polypeptide of Mr 8 kDa was purified from the venom of the South American 'armed' or wandering spider Phoneutria nigriventer by centrifugation, gel filtration on Superose 12, and reverse phase FPLC on columns of Pharmacia PepRPC and ProRPC. The purified neurotoxin Tx1 had an LD50 of 0.05 mg/kg in mice following intracerebroventricular injection. The complete amino acid sequence of the neurotoxin was determined by automated Edman degradation of the native and S-carboxymethylated protein in pulsed liquid and dual phase sequencers, and by the manual DABITC/PITC double coupling method applied to fragments obtained after digestions with the S. aureus V8 protease and trypsin. The neurotoxin Tx1 consists of a single chain of 77 amino acid residues, which contains a high proportion of cysteine. The primary structure showed no homology to other identified spider toxins.