Physico-chemical characterization of gamma rays irradiated crotamine

Abstract

Ionizing radiation can change the molecular structure and affect the biological properties of biomolecules. It has been employed to attenuate animal toxins. Crotamine, a toxin from Crotalus durissus terrificus (Cdt), is a highly basic polypeptide (pI - 10.3), with myotoxic activity and molecular weight of 4882 Da. It is composed of 42 amino acids residues and reticulated by three disulfide bonds. This study aimed the characterization of irradiated crotamine using Circular Dichroism (CD), Fluorescence Spectroscopy and Differential Scanning Calorimetry (DSC) techniques. We used size exclusion and ion-exchange chromatography to purify it from Cdt crude venom. The pure crotamine was irradiated with 2.0 kGy from a 60Co source. Native and irradiated crotamine were analyzed in a fluorescence spectrophotometer (Hitachi F-4500), under excitation wavelength at 275 nm and the emission was scanned from 300 to 500 nm. The analysis of fluorescence quenching showed that the irradiated form displayed a lower quantum yield when compared to the native form. CD spectra, obtained from a Jasco, J-180 spectropolarimeter, of native and irradiated crotamine solutions, showed a discrete change between the samples, from apparently ordered conformation to a random coil. Finally, the thermodynamics analysis, realized in a calorimeter METTLER TOLEDO, DSC 822e, showed that irradiation promoted changes in the calorimetric profile. Our results indicate that irradiation leads to progressive changes in the structure of the toxin, which could explain the decrease in myotoxic activity.