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Characterization of the peptide content in snake venoms can be an important tool for the investigation of new pharmacological lead compounds. For this purpose, single-step analysis of crude venoms has recently been demonstrated using mass spectrometry (MS) techniques. Reproducible profiles of ions in MS and MS/MS spectra may also be used to compare venoms from different species. In this work matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was used to obtain mass patterns of the major peptides (<8 kDa) found in pooled venoms from the genera Bothrops and Crotalus. Venoms from five different Bothrops species (B. jararaca, B. insularis, B. alternatus, B. jararacussu, and B. neuwiedi) and three Crotalus species (C. viridis, C. adamanteus and C. durissus terrificus) were analyzed. In agreement with other reports, venoms from Bothrops species contained a variety of peptides in the range m/z 1000-1500, and in some samples larger components (m/z 7000-8000) were detected. In the Crotalus species venoms were rich in peptides ranging from m/z 1000-1500 and 4000-5500. MS/MS experiments on the low molecular mass peptides (m/z 1000-1500) confirmed the presence of ten new bradykinin-potentiating peptides among venoms from genera Bothrops and Crotalus. In order to determine whether additional peptides could be identified after partial purification, B. jararaca venom was subjected to size-exclusion chromatography on Sephacryl S-200, and two distinct low molecular mass pools were analyzed further by MALDI-TOFMS. No additional peptides were detected from the pool with masses below 2000 Da but a substantial improvement with better resolution was observed for the pool with masses above 7000 Da, indicating that complex samples such as crude snake venoms can be analyzed for low molecular mass peptides using a single-step procedure.

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ABSTRACT.Bothrops moojeni is an abundant venomous snake responsible for most of the snakebite cases in the Central region of Brazil and as a result of the anthropogenic habitat disturbance, such as the increase in extensive farming, the range of B. moojeni has been greatly fragmented. Here, we obtained genomic DNA from a total of 75 snakes belonging to four populations. Genetic variability evaluated for five RAPD primers was low (He = 0.20) and was not spatially structured. We found evidence of significant genetic divergence among B. moojeni populations that were isolated (Φ ST values of 0.21 and 0.25), while populations more proximal exhibited less divergence (Φ ST values of 0.04 and 0.08). We found only moderate divergence (Φ ST value of 0.12) between two populations greatly isolated (851.83 km apart) along with great differentiation (0.24) between two proximal populations (290 km apart). Even though these populations are close to each other, they occur in an urbanized area that is almost completely covered by extensive crops, representing an obstruc- tion to the mobility of this viper. Molecular variance analysis (AMOVA) showed some degree of subdivision in these populations, with a Φ ST value of 0.16, significant to the level of 1% by 1000 random permutations. We also performed a Bayesian analysis that confirmed the AMOVA results and found a value of θ B = 0.14 and an ƒ = 0.27, suggesting a high level of endogamy. This is the first study that characterizes genetic variability for this important species of the Bothrops genus, and our data are of significant importance in terms of classifying populations in relation to their conservational value and management strategies. Thus, given the high levels of population structure found in this case, we recommend sampling as many populations as possible to maximize the genetic variability to be preserved when aiming for in situ conservation. The same should be done to perform samplings toward ex situ conservation.