New insights into the structural characteristics of irradiated crotamine

Abstract

BackgroundSince ionizing radiation has the potential to alter the molecular structure and affect the biological properties of biomolecules, it has been successfully employed to attenuate animal toxins. The present study aimed to characterize the structural modifications on irradiated crotamine, a toxin from Crotalus durissus terrificus venom, using circular dichroism (CD), fluorescence, Fourier transformed infrared spectroscopy (FTIR), atomic force microscopy (AFM) and differential scanning calorimetry (DSC).MethodsA combination of size exclusion and ion-exchange chromatography was used to purify the peptide using crude venom. The pure toxin was then submitted to 2 kGy gamma irradiation doses from a cobalt-60 source. Native and irradiated crotamine were analyzed using a fluorescence spectrophotometer. Wavelength was fixed at 295 nm and fluorescence emission scans were collected from 300 to 400 nm. CD and FTIR techniques were used to identify the secondary structure of both samples. DSC analyses were performed at a starting temperature of 20 °C up to a final temperature of 90 °C. AFM provided a 3D profile of the surfaces of both crotamine forms adsorbed on mica.ResultsFluorescence spectroscopy showed that the quantum yield of the irradiated form decreased. CD spectra of native and irradiated crotamine solutions showed differences between the samples in wavelength, indicating that irradiation induced a transition of a small portion of the random coil regions towards an α-helical conformation. FTIR and CD showed that the native and irradiated crotamine spectra were different with regard to secondary structure. The thermodynamic analysis showed that irradiation caused changes in the calorimetric profile and CD showed that temperature-induced changes also occur in the secondary structure. Finally, AFM showed the possible formation of insoluble aggregates.ConclusionsOur results indicate that irradiation leads to progressive changes in the structure of the toxin, which could explain a decrease in myotoxic activity.

A Fourier Transform Infrared Spectroscopic Investigation of Macrovipera lebetina lebetina and M. l. obtusa Crude Venoms

“…In the field of toxinology, FTIR spectroscopy has been used to analyze the secondary structure of purified snake venom proteins (30)(31)(32)(33) but there is scarce data on the physicochemical characterization of crude snake venoms. In one study, the influence of ecological factors on the venom of M. l. obtusa (from Azerbaijan) was investigated by radiobiological and biophysical (spectroscopic) methods (37).…”

“…The Fourier Transform Infrared Spectroscopy method was used to analyze the secondary structures of purified snake venom toxins (30)(31)(32)(33) and their complexes with nanomaterials (34,35). In one study, the FTIR spectrum of Echis carinatus venom was published together with its chitosan nanoparticle complex but the results were used for comparative purposes to assess the loading efficiency and it was not aimed to make a venomic characterization (36).…”

Comparação entre a nefrotoxicidade da crotoxina nativa e a irradiada com raios gama de Co-60 em camundongos

“…Já se sabe que a irradiação de toxinas com raios gama que resulta em mudanças estruturais e consequentemente diminuem as atividades enzimáticas de proteínas tóxicas sem, entretanto, comprometer os epítopos imunogênicos (OLIVEIRA et al, 2015).…”

Resposta imunológica em modelos animais imunizados contra o muco nativo ou irradiado por raios gama de ⁶⁰Co da raia de água doce <i>Paratrygon aiereba</i>

“…A partir da realização desses ensaios foi possível verificar que tanto o muco quanto o veneno de Paratrygon aiereba são antigênicos, estimularam a produção de anticorpos, e que esta capacidade antigênica provavelmente se deve ao fato que venenos animais, inclusive de peixes, são compostos por uma variedade de proteínas e peptídeos biologicamente ativos (Conceição et al, 2006;2011;Saraiva et al, 2011;Komegae et al, 2013;Ziegman;Alewood, 2015 Normalmente, coelhos são utilizados como modelos experimentais para a produção de antissoro policlonal por serem bons respondedores imunológicos (Burns, 2005). E diferenças da resposta imune entre diferentes modelos experimentais foram observadas por Martín e colaboradores 2015 A irradiação do muco por raios gama de 60 Co justifica-se a partir de trabalhos publicados anteriormente por nosso grupo, demonstrando que as radiações ionizantes promovem alterações físico-químicas nas moléculas de proteínas de venenos de serpentes e toxinas isoladas, atenuando a atividade proteolítica de toxinas e mantendo suas propriedades imunológicas (Nascimento et al, 1996;Boni-Mitake et al, 2001;Camilo et al, 2005;Alves, 2009;Caproni et al, 2009;Ferreira Jr. et al, 2009;Oliveira et al, 2015;Rocha, 2016).…”