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

Abstract

Background: 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.

Keywords: Enzymatic activity; Fragmentation; Gamma rays; Radiation.

Figure 1.. SDS-PAGE under non-reducing conditions (7.5% polyacrylamide gel, Mini Protean II System, Bio-Rad) of (A) native TeNT and TeNT irradiated with (B) 1 kGy, (C) 2 kGy, (D) 3 kGy, (E) 4 kGy, (F) 5 kGy, (G) 6 kGy, (H) 7 kGy and (I) 8 kGy. (MW) Molecular mass markers (Precision Plus Protein^TM Standards Bio Rad: mixture of 10 recombinant proteins, 10-250 kDa, 8 blue-stained bands and 2 pink reference bands - 25 and 75 kDa). Arrows point to tetanus toxin (150 kDa).

Figure 2.. SDS-PAGE under reducing conditions (7.5% polyacrylamide gel, Mini Protean II System, Bio-Rad) of (A) native TeNT and TeNT irradiated with (B) 1 kGy, (C) 2 kGy, (D) 3 kGy, (E) 4 kGy; (F) 5 kGy; (G) 6 kGy, (H)7 kGy and (I)8 kGy. (MW) Molecular mass markers (Precision Plus Protein^TM Standards Bio Rad: mixture of 10 recombinant proteins, 10-250 kDa, 8 blue-stained bands and 2 pink reference bands - 25 and 75 kDa). Arrows point to tetanus toxin 100 kDa (heavy chain) and 50 kDa (light chain).

Figure 3.. Determination of peptide number nTeNT and iTeNT with 1 kGy, 2 kGy, 3 kGy, 4 kGy, 5 kGy, 6 kGy, 7 kGy and 8 kGy by liquid chromatography-mass spectrometry (LC-MS), using an electrospray-ion trap-time of flight (ESI-IT-TOF) system coupled to a binary ultra-fast liquid chromatography system (UFLC) (20 A Prominence, ShimadzuKyoto, Japan).

Figure 4.. Mass spectra profiles of (A) native TeNT and TeNT irradiated with (B) 1 kGy, (C) 2 kGy, (D) 3 kGy, (E) 4 kGy, (F) 5 kGy, (G) 6 kGy, (H) 7 kGy, (I) 8 kGy by matrix associated laser desorption ionization-time of flight MALDI-TOF/TOF (Axima Performance, Shimadzu®) mass spectrometer.

Figure 5.. Antigenic characteristics of nTeNT and iTeNT was evaluated by ELISA using sera of immunized mice with Td vaccine. Results are presented as a binding of TeNT-specific-Abs to the same amount of nTeNT and iTeNTs. Samples are assessed in hexaplicates and results presented as mean ± SE. The statistical significance of the observed differences in binding of a given Abs to nTeNT and to iTeNT was calculated using Student’s t-test (*p < 0.05, **p < 0.005, ***p < 0.001).

Figure 6.. Western blot analysis of reactivity of TeNT-specific Abs toward H and L chain of TeNT. nTeNT and iTeNT was resolved on 7.5% polyacrylamide gel by SDS-PAGE under reducing conditions: (A) native; (B) 1 kGy; (C) 2 kGy; (D) 3 kGy; (E) 4 kGy; (F) 5 kGy; (G) 6 kGy; (H) 7 kGy; (I) 8 kGy.

Figure 7.. Enzymatic activity of nTeNT and iTeNT by FRET: Abz-GASQ↓FETSA-Q-EDDnp. Abz is bound to the N-amino terminal of synaptobrevin (aminoacids residues 73-81) and EDDnp to the C-terminal carboxyl group. Arrow (↓) indicates the bond cleaved.