Purification and Biochemical Characterization of TsMS 3 and TsMS 4: Neuropeptide-Degrading Metallopeptidases in the Tityus serrulatus Venom
Abstract
:1. Introduction
2. Results
2.1. Isolation of TsMS 3 and TsMS 4
2.2. Bioinformatic Analysis
2.3. Comparative Analysis of Metalloserrulases Biochemical Parameters
2.4. Determination of Cleavage Sites
2.5. Kinetic Parameters Determination
2.6. In Vitro Neutralization Assay of the Activity of Metalloserrulases by Commercial Antivenoms
3. Discussion
4. Conclusions
5. Materials and Methods
5.1. Reagents
5.2. Venoms and Antivenoms
5.3. Purification of Metalloserrulases 3 and 4 from Tityus serrulatus Venom
5.3.1. Chromatographic Steps
5.3.2. Screening Using FRET Substrate
5.3.3. Screening Using Human Neuropeptides
5.4. Characterization of Isolated Proteases
5.4.1. SDS-PAGE—In Gel Digestion and Mass Spectrometry
5.4.2. Biochemical Characterization: Effect of pH, Cation Concentration and Temperature
5.4.3. Analysis of The Cleavage Sites
5.4.4. Kinetic Parameters for The Hydrolysis of FRET Substrates by TsMS 4
5.5. Sera Neutralization Assays
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Step | Fraction | Volume (µL) | Total Protein (µg) | Activity (units) | Total Activity (units/µL) | Specific Activity (units/µg) | Purification Factor | Yield (%) | EDTA Inhibition (%) |
---|---|---|---|---|---|---|---|---|---|
Venom | TsV | 1000 | 30,000 | 90 | 90,000 | 3000 | 1 | 100 | 100 |
DEAE | F3 | 1000 | 0.12 | 13.1 | 13,100 | 109,167 | 36.39 | 14.6 | 100 |
F5 | 1000 | 0.139 | 20.9 | 20,873 | 150,165 | 50.06 | 23.2 | 100 | |
GF | F3-4 | 1000 | 0.024 | 8.9 | 8900 | 370,833 | 123.61 | 9.9 | 100 |
F5-1 | 1000 | 0.071 | 12.7 | 12,729 | 179,276 | 59.76 | 14.1 | 100 |
Peptide | Specific Activity (µM/µg/min) | Scissile Bonds | |
---|---|---|---|
TsMS 3 | TsMS 4 | ||
NPY | 0.433 | 0.631 | YPSKP↑D↓NPGED↓↑A↑PAEDM↓A↓↑R↓↑Y↓↑Y↓↑S↓A↓L↓↑R↓↑H↓↑Y↑I↓N↓↑LIT↓R↓↑Q↓↑RY-NH2 |
PYY | 0.359 | 0.420 | YPIKPEAPGED↓ASPEEL↓N↓R↓YYASL↓↑R↓HY↓L↓NLVT↑R↓Q↓RY-NH2 |
PP | 0.347 | 0.430 | APLEPMYP↓G↑D↑Y↑A↑TH↑E↑Q↑RAQ↑YETQL↓↑R↑R↓↑Y↑YPIKPE↓↑PRY-NH2 |
DYN | 1.539 | 1.303 | YGGFL↑R↓↑RIRPKLK |
Substrate | Catalytic Constants | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
P4 | P3 | P2 | P1 | P1′ | P2′ | Km (µM) | kcat (s−1) | kcat/Km (µM−1·s−1) | ||
Abz | G | F | L | R | R | V | EDDnp | 16.2 ± 4.3 | 491.7 ± 52.4 | 30.4 ± 8.1 |
Abz | G | F | L | R | R | - | EDDnp | 36.6 ± 5.8 | 170.8 ± 8.4 | 4.7 ± 0.7 |
Abz | - | F | L | R | R | V | EDDnp | 28.1 ± 2.6 | 1108.3 ± 33.2 | 39.4 ± 3.64 |
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Cajado-Carvalho, D.; da Silva, C.C.F.; Kodama, R.T.; Mariano, D.O.C.; Pimenta, D.C.; Duzzi, B.; Kuniyoshi, A.K.; Portaro, F.V. Purification and Biochemical Characterization of TsMS 3 and TsMS 4: Neuropeptide-Degrading Metallopeptidases in the Tityus serrulatus Venom. Toxins 2019, 11, 194. https://doi.org/10.3390/toxins11040194
Cajado-Carvalho D, da Silva CCF, Kodama RT, Mariano DOC, Pimenta DC, Duzzi B, Kuniyoshi AK, Portaro FV. Purification and Biochemical Characterization of TsMS 3 and TsMS 4: Neuropeptide-Degrading Metallopeptidases in the Tityus serrulatus Venom. Toxins. 2019; 11(4):194. https://doi.org/10.3390/toxins11040194
Chicago/Turabian StyleCajado-Carvalho, Daniela, Cristiane Castilho Fernandes da Silva, Roberto Tadashi Kodama, Douglas Oscar Ceolin Mariano, Daniel Carvalho Pimenta, Bruno Duzzi, Alexandre Kazuo Kuniyoshi, and Fernanda Vieira Portaro. 2019. "Purification and Biochemical Characterization of TsMS 3 and TsMS 4: Neuropeptide-Degrading Metallopeptidases in the Tityus serrulatus Venom" Toxins 11, no. 4: 194. https://doi.org/10.3390/toxins11040194
APA StyleCajado-Carvalho, D., da Silva, C. C. F., Kodama, R. T., Mariano, D. O. C., Pimenta, D. C., Duzzi, B., Kuniyoshi, A. K., & Portaro, F. V. (2019). Purification and Biochemical Characterization of TsMS 3 and TsMS 4: Neuropeptide-Degrading Metallopeptidases in the Tityus serrulatus Venom. Toxins, 11(4), 194. https://doi.org/10.3390/toxins11040194