9 Da), both with amidated

9 Da), both with amidated find more C-terminal, and endowed with antimicrobial and hemolytic activities [8] and [9]. In 2004, Yamaji and colleagues [31] described IsTX, a sex-specific α-KTX toxin, exclusively found in the venom of O. madagascariensis males. IsTX (α-KTx 6.11) has 41 residues with the conserved CS-αβ structure stabilized by four disulfide bridges. IsTX shows high affinity toward both voltage and Ca2+-activated K+ channels. Additionally, IsTx presents great similarity with HsTX1 (α-KTx 6.3), a toxin isolated from Heterometrus spinifer [16] that blocks Kv1.3 channels with picomolar affinity [31]. In Brazil, the genus Opisthacanthus has two

species: O. borboremai [17] and O. cayaporum. O. cayaporum has no medical significance, and up to date there are only three studies published concerning this species: the proteomics [25] and transcriptome [27] of the venom gland, and the description of κ-KTx 2.5, an inhibitor of hKv1.1, and hKv1.4 channels, having a higher affinity for Kv1.4 (IC50 = 71 μM) [4]. Here we describe the purification and functional characterization of a toxin denoted by the trivial name as OcyKTx2, which stands for KTx 2 from the venom of O. cayaporum. This peptide falls into subfamily 6 of the α-KTx scorpion toxins (systematic name, α-KTx6.17). OcyKTx2 is a 34 amino acid long peptide with four disulfide bridges and molecular mass of 3807 Da

that reversibly blocks Shaker B K+-channels with a Kd of 82 nM, and presents an even better affinity toward Kv1.3, blocking it with a Kd of ∼18 nM. Individuals of O. cayaporum of both sexes, collected in Palmas, in the State of Tocantins, Brazil, under license from IBAMA 048/2007-CGFAU, I-BET-762 manufacturer were kept in appropriate terrariums in the Laboratory of Toxicology at the University of Brasilia, where they received water ad libitum and were fed periodically with cockroaches. anti-PD-1 monoclonal antibody Crude venom was obtained by electrical stimulation. The material was extracted with water and centrifuged at 10,000 × g for 10 min. The soluble supernatant was stored at −20 °C and separated by high performance liquid chromatography (HPLC) in a C18 reverse-phase

analytical column (250 mm × 4.60 mm, 4 μ, Phenomenex, Inc., USA), using a linear gradient from 0% solvent A (0.12% trifluoroacetic acid, TFA, in water) to 60% solvent B (0.10% TFA in acetonitrile) in 60 min, at a flow rate of 1 mL/min. The fraction of interest was pooled and further purified on the same column in order to obtain OcyKTx2 in homogeneous form. Amino acid sequence determination of OcyKTx2 was performed by automatic Edman degradation in a Beckman LF 3000 Protein Sequencer (Palo Alto, CA, USA), after adsorption of the sample into CD-Inmmobilon membranes, as described by the manufacturer. The OcyKTx2 was reconstituted in 50% acetonitrile with 1% acetic acid and directly applied into a Finnigan LCQDUO ion trap mass spectrometer (San Jose, CA) using a Surveyor MS syringe pump delivery system and a C18 PicoFrit column/needle (75 mm × 10.

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