The theoretical physical–chemical parameters were predicted and analyzed by ProtParam (www.expasy.org). The comparison of the amino acid sequence with other LmLAAO was performed using FASTA (http://www.ebi.ac.uk/Tools/fasta/index.html) and BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). In order to predict the three-dimensional structure of LmLAAO, a model based on sequence homology was manually built. Search for protein homologues was initially performed by using the BLAST search algorithm (http://www.ncbi.nlm.nih.gov) against the protein data bank (www.rcsb.org). The crystallographic structure of l-amino acid oxidase from Agkistrodon

halys pallas (PDB:1REO) ( Zhang et al., 2004), which shares 90% of sequence identity with LmLAAO, has been identified and used as a template for molecular modeling. Based on the sequence alignment performed by Multalin ( Corpet, 1988), amino acid substitutions selleck compound were manually included LY294002 in the model by using COOT ( Emsley and Cowtan, 2004). Structure refinement was performed using molecular dynamics with simulated annealing in CNS ( Brunger et al., 1998). Stereochemistry of the predicted model was checked by PROCHECK ( Laskowski et al., 1993). Animal care was in accordance with ethical recommendations of the International Guiding Principles for Biomedical Research Involving Animals of the Council of International Organizations of Medical

Sciences (CIOMS) and was approved by the Institutional Committee for the Care and Use of Laboratory Animals (CICUA) of the University of Costa Rica (no CICUA-012-08). The hemorrhagic activity was determined by intradermal injection of 50 μg of LmLAAO dissolved in 50 μL of PBS solution in the abdominal region of three mice (strain CD-1, 18–22 g). After 3 h, mice were sacrificed, and the skin was observed for

hemorrhagic halo formation (Gutiérrez et al., 1985). A group of 6 mice (CD-1, 18–22 g) were injected with 10 μg LmLAAO subcutaneously in the subplantar region of the right footpad. The left footpad was used as control and injected with PBS. At different time intervals (15 min, 1, 3 and 24 h), the thickness of the mice paws was measured with a low-pressure spring caliper (Lomonte et al., 1993). The formation selleckchem of edema was expressed as a percentage of increment in footpad thickness. The systemic toxicity was evaluated in a group of 6 mice (CD-1, 18–22 g) by intravenous injection of 100 μg of LmLAAO. A group of 6 mice (CD-1, 18–22 g) injected with PBS was used as control. Animal behavior was monitored during the first 3 h after injection. After 24 h of injection, animals were sacrificed and the tissues of heart, lung and kidney were removed, dissected, and samples were processed for histological observation, embedded in paraffin, cut to 4 mm thick and stained with hematoxylin and eosin. A group of 5 mice (CD-1, 18–22 g) was injected intramuscularly in the right quadriceps with a solution containing 100 μg of LmLAAO dissolved in PBS.