Both samples were loaded in a Phenomenex C18 column (Jupiter 5 μ, 4 × 150 mm, California, USA) in a two-solvent system: (A)

trifluoroacetic acid (TFA)/H2O (1:1000) and (B) TFA/Acetonitrile (ACN)/H2O (1:900:100). The column was eluted at a flow rate of 1 mL/min with a 10–80% gradient of solvent B over 40 min. The HPLC column eluates were monitored by their absorbance at 214 nm. The peptides eluted were analyzed on a MALDI-ToF/PRO instrument (G&E Healthcare – Sweden). Samples were mixed 1:1 (v: v) with a supersaturated solution matrix for peptides (α-cyano 4-hydroxycinnamic acid in 50% acetonitrile containing 0.1% TFA), deposited on the sampling plate (0.4–0.8 l) and dried. The spectrometer was operated in reflectron mode and P14R ([M + H+] + 1533.85) and angiotensin II ([M + H+] + 1046.54)

(Sigma, St. Louis, MO) were used as external calibrants. SDS-PAGE was carried out according to the method of Laemmli (1970). Sting selleck venom, skin mucus and protein fractions (10 μg) of C. spixii were analyzed by SDS-PAGE JAK inhibition 4–20% acrylamide gradient under reducing conditions. Prior to electrophoresis, the samples were mixed 1:1 (v/v) with sample buffer. The gel was stained with the Silver method. For protein deglycosylation under denaturing conditions, toxin samples (20 μg) were incubated in 10% SDS for 1 min at 95 °C. After adding 0.02 M sodium phosphate buffer, 0.08% sodium azide, 0.01 M EDTA, 2% Triton X-100, pH 7.0, incubation was prolonged for 2 min at 95 °C. After cooling, 1 U of N-glycosidase F (Roche, Mannheim, Germany) was added, and the mixture was incubated for 1 h at 37 °C. The deglycosylation profiles were evaluated by SDS-PAGE as described above. The protein Fv6 was reduced and alkylated with 4-vinyl pyridine as described (Wilson and Yuan,

1989). One milligram-aliquots of Fv6 were dissolved in 1 ml of 0.1 M Tris–HCl (pH 8.6), 6 M guanidine-HCl. After addition of 30 μL β-mercaptoethanol the samples were incubated first at 50 °C for 4 h under nitrogen, then after addition of 40 μL of 4-vinyl pyridine, in the dark at 37 °C for 2 h and subsequently desalted on a PD-minitrap G25 column. The S-pyridylethylated proteins were cleaved with 2% (w/w) chymotrypsin at 37 °C for 3 h. The cleavage products were separated on a Vydac C18 small pore column (4.6 × 250 mm) Fossariinae in a linear gradient of 0–50% acetonitrile in 0.1% aqueous TFA and sequenced using a Shimadzu PPSQ-21A protein sequencer. The partial primary structure of Fv6 was compared with the sequences of other related proteins in the SWISS-PROT/TREMBL data bases using the FASTA 3 and BLAST programs. The dynamics of alterations in the microcirculatory network were determined using intravital microscopy by transillumination of mice cremaster muscle after subcutaneous application of 10 μg of all fractions, sting venom or skin mucus of C. spixii dissolved in 20 μL of sterile saline. Administration of the same amount of sterile saline was used as control.