Action of Bromelain and Ficin on horse anti Bothrops sp venom Antibodies

Abstract The treatment with hyperimmune sera constitute the only specific and effective therapy available against snakebite envenomation, most common in developing countries. Serum quality is an important factor on patient recovery time and in the incidence of death and permanent disability. To date, most sera consist of pepsin digested IgG antibodies harvested from hyperimmune animals. The use of animal derived enzymes, such as pepsin, to digest IgG, constitute a source of adventitious agents and contaminants, such as porcine circovirus. The present study aims to evaluate the use of the plant derived enzymes bromelain and ficin, as an alternative to pepsin. To this purpose, horse serum immunized against Bothrops venoms was purified with caprylic acid and digested with bromelain or ficin. SDS-PAGE results evidence the formation of F(ab)'2 fragments and suggest that a digestion time superior to 8 hours may be required to completely digest the antibodies with bromelain or ficin. F(ab)'2 fragments obtained by digestion with either bromelain or ficin digestion preserved the ability to recognize Bothrops sp. venom in western blotting assays. Therefore, both enzymes are suitable for use in large-scale production, minimizing contamination risks and increasing safety and efficiency of serotherapy treatments.

Characterization and evaluation of the enzymatic activity of tetanus toxin submitted to cobalt-60 gamma radiation

Tetanus toxin blocks the release of the inhibitory neurotransmitters in the central nervous system and causes tetanus and its main form of prevention is through vaccination. The vaccine is produced by inactivation of tetanus toxin with formaldehyde, which may cause side effects. An alternative way is the use of ionizing radiation for inactivation of the toxin and also to improve the potential immunogenic response and to reduce the post-vaccination side effects. Therefore, the aim of this study was to characterize the tetanus toxin structure after different doses of ionizing radiation of 60Co. Methods Irradiated and native tetanus toxin was characterized by SDS PAGE in reducing and non-reducing conditions and MALD-TOF. Enzymatic activity was measured by FRET substrate. Also, antigenic properties were assessed by ELISA and Western Blot data. Results Characterization analysis revealed gradual modification on the tetanus toxin structure according to doses increase. Also, fragmentation and possible aggregations of the protein fragments were observed in higher doses. In the analysis of peptide preservation by enzymatic digestion and mass spectrometry, there was a slight modification in the identification up to the dose of 4 kGy. At subsequent doses, peptide identification was minimal. The analysis of the enzymatic activity by fluorescence showed 35 % attenuation in the activity even at higher doses. In the antigenic evaluation, anti-tetanus toxin antibodies were detected against the irradiated toxins at the different doses, with a gradual decrease as the dose increased, but remaining at satisfactory levels. Conclusion Ionizing radiation promoted structural changes in the tetanus toxin such as fragmentation and/or aggregation and attenuation of enzymatic activity as the dose increased, but antigenic recognition of the toxin remained at good levels indicating its possible use as an immunogen. However, studies of enzymatic activity of tetanus toxin irradiated with doses above 8 kGy should be further analyzed.(AU)

Protein identification from the parotoid macrogland secretion of Duttaphrynus melanostictus

Bufonid parotoid macrogland secretion contains several low molecular mass molecules, such as alkaloids and steroids. Nevertheless, its protein content is poorly understood. Herein, we applied a sample preparation methodology that allows the analysis of viscous matrices in order to examine its proteins. Methods: Duttaphrynus melanostictus parotoid macrogland secretion was submitted to ion-exchange batch sample preparation, yielding two fractions: salt-displaced fraction and acid-displaced fraction. Each sample was then fractionated by anionic-exchange chromatography, followed by in-solution proteomic analysis. Results: Forty-two proteins could be identified, such as acyl-CoA-binding protein, alcohol dehydrogenase, calmodulin, galectin and histone. Moreover, de novo analyses yielded 153 peptides, whereas BLAST analyses corroborated some of the proteomic-identified proteins. Furthermore, the de novo peptide analyses indicate the presence of proteins related to apoptosis, cellular structure, catalysis and transport processes. Conclusions: Proper sample preparation allowed the proteomic and de novo identification of different proteins in the D. melanostictus parotoid macrogland secretion. These results may increase the knowledge about the universe of molecules that compose amphibian skin secretion, as well as to understand their biological/physiological role in the granular gland.(AU)

New insights into the structural characteristics of irradiated crotamine

Background:Since ionizing radiation has the potential to alter the molecular structure and affect the biologica 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).Methods:A 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.Results:Fluorescence 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 a-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.Conclusions:Our results indicate that irradiation leads to progressive changes in the structure of the toxin, which could explain a decrease in myotoxic activity.(AU)

New insights into the structural characteristics of irradiated crotamine

Background: Since ionizing radiation has the potential to alter the molecular structure and affect the biologica 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). Methods: A 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. Results: Fluorescence 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 a-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. Conclusions: Our results indicate that irradiation leads to progressive changes in the structure of the toxin, which could explain a decrease in myotoxic activity.