On the other hand, knowledge regarding the composition of Crotalus oreganus abyssus venom is scarce [24]. From previous experiments in our laboratory in which we have studied the effects of peptides isolated from C. o. abyssus venom, we showed the presence of a natriuretic peptide in its venom (now called Coa_NP1) that produced hypotensive and vasorelaxation effects [5]. The aim of the present study was to identify and investigate the systemic and vascular effects of a new natriuretic peptide isolated from C. o. abyssus venom (Coa_NP2). All reagents were purchased from Aldrich

or Sigma Co. (USA). C. o. abyssus (Coa) venom was obtained from The National Natural Toxins Research Center (NNTRC) of Texas A&M University-Kingsville (Kingsville, TX, USA). C. o. abyssus (Coa) whole venom was submitted to selleck kinase inhibitor an FPLC molecular exclusion chromatographic column packed with Sephadex 75 (Akta Primer, GE, USA), exactly as described in [5]. In this separation, five peaks were observed (I–V) ( Fig. 1A). Only fraction V presented a hypotensive effect. To perform a better study and for separation

of peptides and proteins, fraction Silmitasertib V was then submitted to ultra-filtration using the MidJet apparatus (Ge Healthcare, USA), equipped with the UFP-10-C-MM01A cartridge, and a superficial area of 26 cm2, cut off: 10,000 Da (Ge Healthcare, USA). The filtrate presented hypotensive effects and was lyophilized and stored at −20 °C, until use. The filtrate was subjected to reverse phase HPLC (model 2010, Shimadzu, Japan) using an analytical C5 column (Supelco, 250 mm × 4.6 mm), which was previously equilibrated with buffer A (0.1% TFA). The filtrate (10 mg) were dissolved completely in buffer A (0.1% TFA), centrifuged at 5000 × g and then loaded onto a reverse-phase column. The peptides were purified using a linear gradient of buffer B concentration (66% acetonitrile in buffer A) and the chromatographic UV monitoring was carried out at 216 nm [6] and [7]. For electrophoresis, Tricine PAGE-SDS Adenosine triphosphate was used for characterization of low molecular weight proteins

and peptides [32]. Each peak or peak group was tested for its action on blood pressure and Coa_NP2 showed positive results. Two milligrams of the purified peptide were dissolved in 200 μl of a 6 mol/l guanidine chloride solution containing 0.4 mol/l of Tris–HCl and 2 mmol/l EDTA (pH 8.15). Nitrogen was blown over the top of the protein solution for 15 min, before reducing with DTT (6 M, 200 μl). This solution was incubated in the dark at 37 °C for 1 h and desalted using a Sephadex G25 column (0.7 cm × 12 cm) with 1 mol/l acetic acid buffer. The reduced peptide was sequenced using an automatic peptide sequencer (890C automatic sequencer, Beckman, USA). The phenylthyoidantoin (PTH) amino acids were identified by comparing their retention times to the 20 PTH amino acid standards [2].