The chromatographies were done using a Shimadzu 20A

HPLC modular equipment with an SPD-20A detector, selleck chemical and the data were generated and stored using the Shimadzu LC-Solutions Software, acquiring the absorbance data at a rate of 1 Hz, resulting in a total of 9005 points which were processed for each chromatography. The fractal dimension (D) analysis on the chromatographic profiles of the venoms was calculated for the initial 60 min of venom fractioning. This analyses is an alternative to study inter and intraspecific venom variability taking advantage of the multiple waveforms of these and comparing them point to point in all or in partial intervals of elution time. By this study, it is also possible to calculate the probability (P) of the difference Cabozantinib clinical trial between two values of D (ΔD) that is used to indicate the contortedness of waveforms, inter venoms. The data were transformed to ASCII format (American

Standard Code for Information Interchange) in data pairs, and analyzed under VeFractDim software according to D’Suze and Sevcik (2010). For the phase plot of D analysis a sliding window sequences (SWS) of the 500 continuous points starting from time 0 of the chromatography was used and with a recursive displacing by 1 s for 60 min of elution stored as [(ti,Di) sets]. For this data sets the contortedness represented by Q = D − 1 was calculated, and a phase plot with their (ti,Qi) sets was constructed. The determination coefficient (ds) was calculated squaring the Spearman rank correlation coefficient as suggested previously ( D’Suze and Sevcik, 2010). The data were plotted using the GraphPad Prism 5 software.

Phosphoribosylglycinamide formyltransferase All fractions obtained from Ts-DF and Ts-MG venom chromatography separation were analyzed by mass spectrometry performed on a MALDI-TOF AutoFlex III (Bruker Daltonics®, Germany) in linear and reflector modes and the spectra were processed with MassLynx™3.5 (Manchester, UK) and FlexAnalysis 3.3 (Bruker Daltonics®, Germany). Briefly, solubilized fractions (0.5 μL of sample, variable concentrations) were spotted onto the target followed by 0.5 μL of CHCA (α-cyano-4-hidroxycinnamic acid) matrix solution (60% acetonitrile/0.3% TFA), and allowed to dry at room temperature (dried-droplet method). Peptide Calibration Standard II (700 − 4000 Da) and Protein Calibration Standard I (3000–25,000 Da) (Bruker Daltonics®, Germany) were used as external calibrates. Mass spectra from the average of 256 laser pulses from m/z 600 to 39,400 were obtained. The experimental procedure was approved by the Ethical Committee for Animal Experimentation of Brasilia University (CEUA/UnB) under protocol number 133424/2009.