Identification of crotamine in the venom of Crotalus durissus collilineatus by three different methods.

The use of mice for the identification of crotamine has been the motive of discussions of bioethical character and technical (efficiency), so that a reassessment of the use of animals in experiments is global trend the search for alternative tests. The objective of this study was to standardize a method for HPLC-PDA to identify the presence of crotamine in the venom of rattlesnakes, aiming to propose an alternative methodology to reduce or replace the use of animals. The Cdc was evaluated as to the presence of crotamine by 3 methods: traditional test lethality in mice (Mus musculus) swiss albino male, 18-22 g (i.p.), polyacrylamide gel electrophoresis (SDS-PAGE) and HPLC-PDA. The venoms of 50 specimens of Crotalus durissus collilineatus, held in the serpentarium CEPB/PUC Goiás, were obtained by manual massage of the gland, making the collection individually. To identify the band corresponding to crotamine, the venoms of specimens, analysis was performed on SDS-PAGE and references. Procedure in mice with 20% of the samples tested positive for crotamine, 24% negative and 56% uncertain outcome. With the SDS-PAGE was identified crotamine in 26% of samples, 26% negative and 48% continued with uncertain outcome. By HPLC method showed the presence myotoxin in 86% of samples, with 14% negative. The tests conducted in this study indicated that methodology which utilizes animals for identifying the presence of crotamine the venom of C. durissus can safely be replaced by the test SDS-PAGE and HPLC, since the methods are reproducible, and do not undergo any interference biological animal and mainly contribute to reducing the number of animals used for laboratory tests.