The interaction of the antitoxin DM43 with a snake venom metalloproteinase analyzed by mass spectrometry and surface plasmon resonance |
| |
| Authors: | Guilherme D Brand Rune Salbo Thomas J D Jørgensen Carlos Bloch Jr Elisabetta Boeri Erba Carol V Robinson Isabelle Tanjoni Ana M Moura‐da‐Silva Peter Roepstorff Gilberto B Domont Jonas Perales Richard H Valente Ana G C Neves‐Ferreira |
| |
| Institution: | 1. Laboratório de Espectrometria de Massa, Embrapa‐Recursos Genéticos e Biotecnologia, Esta??o Parque Biológico, , 70770‐900 Brasília, DF, Brazil;2. Diabetes Protein Engineering, Novo Nordisk A/S, , DK‐2760 M?l?v, Denmark;3. Department of Biochemistry and Molecular Biology, University of Southern Denmark, , DK‐5230 Odense M, Denmark;4. Laboratory of Organic Chemistry, ETH Zürich, , 8093 Zürich, Switzerland;5. Department of Chemistry, Physical and Theoretical Chemistry Laboratory, University of Oxford, , Oxford, OX1 3QZ United Kingdom;6. Laboratório de Imunopatologia, Instituto Butantan, , Butant?, 05503‐900 S?o Paulo, SP, Brazil;7. Unidade Prote?mica, Laboratório de Química de Proteínas, Instituto de Química, Centro de Tecnologia, Universidade Federal do Rio de Janeiro, , 21941‐909 Rio de Janeiro, RJ, Brazil;8. Rio de Janeiro Proteomic Network/FAPERJ;9. Instituto Nacional de Ciência e Tecnologia em Toxinas/CNPq;10. Laboratório de Toxinologia, Pavilh?o Ozório de Almeida, Instituto Oswaldo Cruz, , 21040‐900 Rio de Janeiro, RJ, Brazil |
| |
| Abstract: | DM43 is a circulating dimeric antitoxin isolated from Didelphis aurita, a South American marsupial naturally immune to snake envenomation. This endogenous inhibitor binds non‐covalently to jararhagin, the main hemorrhagic metalloproteinase from Bothrops jararaca snake venom, and efficiently neutralizes its toxicity. The aim of this study was to apply mass spectrometry (MS) and surface plasmon resonance (SPR) to improve the molecular characterization of this heterocomplex. The stoichiometry of the interaction was confirmed by nanoelectrospray ionization‐quadrupole‐time‐of‐flight MS; from native solution conditions, the complex showed a molecular mass of ~94 kDa, indicating that one molecule of jararhagin (50 kDa) interacts with one monomer of DM43 (43 kDa). Although readily observed in solution, the dimeric structure of the inhibitor was barely preserved in the gas phase. This result suggests that, in contrast to the toxin–antitoxin complex, hydrophobic interactions are the primary driving force for the inhibitor dimerization. For the real‐time interaction analysis, the toxin was captured on a sensor chip derivatized with the anti‐jararhagin monoclonal antibody MAJar 2. The sensorgrams obtained after successive injections of DM43 in a concentration series were globally fitted to a simple bimolecular interaction, yielding the following kinetic rates for the DM43/jararhagin interaction: ka = 3.54 ± 0.03 × 104 M?1 s?1 and kd = 1.16 ± 0.07 × 10?5 s?1, resulting in an equilibrium dissociation constant (KD) of 0.33 ± 0.06 nM. Taken together, MS and SPR results show that DM43 binds to its target toxin with high affinity and constitute the first accurate quantitative study on the extent of the interaction between a natural inhibitor and a metalloproteinase toxin, with unequivocal implications for the use of this kind of molecule as template for the rational development of novel antivenom therapies. Copyright © 2012 John Wiley & Sons, Ltd. |
| |
| Keywords: | metalloproteinase metalloproteinase inhibitor snake venom toxin antitoxin mass spectrometry surface plasmon resonance |
|
|
