BaltDC: purification, characterization and infrared spectroscopy of an antiplatelet DC protein isolated from Bothrops alternatus snake venom

 

Mariana Santos Matias¹, Bruna Barbosa de Sousa^1 8, Déborah Fernanda da Cunha Pereira¹, Edigar Henrique Vaz Dias¹, Carla Cristine Neves Mamede^2 8, Mayara Ribeiro de Queiroz⁸, Anielle Christine Almeida Silva⁴, Noelio Oliveira Dantas⁴, Andreimar Martins Soares^5 6, Júnia de Oliveira Costa^1 7, Fábio de Oliveira^3 8 

 

1 Postgraduate Program in Genetics and Biochemistry, Institute of Genetics and Biochemistry, Federal University of Uberlândia (UFU), Uberlândia, MG, Brazil.

2 Institute of Agricultural Sciences, Federal University of Uberlândia (UFU), Monte Carmelo, MG, Brazil.

3 Institute of Biomedical Sciences, Federal University of Uberlândia (UFU), Uberlândia, MG, Brazil.

4 Institute of Physics, Federal University of Uberlândia (UFU), Uberlândia, MG, Brazil.

5 Center for the Study of Biomolecules Applied to Health (CEBio), Oswaldo Cruz Foundation (Fiocruz – Rondônia) and Health Group, Federal University of Rondônia (UNIR), Porto Velho, RO, Brazil.

6 University Center São Lucas (UniSL), Porto Velho, RO, Brazil.

7 Federal Institute of Education, Science and Technology of Triângulo Mineiro (IFTM), Campus Ituiutaba, Ituiutaba, MG, Brazil.

8 National Institute of Science and Technology in Nanobiopharmaceutics (N-Biofar), Belo Horizonte, MG, Brazil.

 

ABSTRACT

Background:

Snake venoms are a complex mixture of proteins, organic and inorganic compounds. Some of these proteins, enzymatic or non-enzymatic ones, are able to interact with platelet receptors, causing hemostatic disorders. The possible therapeutic potential of toxins with antiplatelet properties may arouse interest in the pharmacological areas. The present study aimed to purify and characterize an antiplatelet DC protein from Bothrops alternatus snake venom.

 

Methods:

The protein, called BaltDC (DC protein from B. alternatus snake venom), was purified by a combination of ion-exchange chromatography on DEAE-Sephacel column and gel filtration on Sephadex G-75. The molecular mass was estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). The amino acid sequence of the N-terminal region was carried out by Edman degradation method. Platelet aggregation assays were performed in human platelet-rich plasma (PRP). Infrared (IR) spectroscopy was used in order to elucidate the interactions between BaltDC and platelet membrane.

 

Results:

BaltDC ran as a single protein band on SDS-PAGE and showed apparent molecular mass of 32 kDa under reducing or non-reducing conditions. The N-terminal region of the purified protein revealed the amino acid sequence IISPPVCGNELLEVGEECDCGTPENCQNECCDA, which showed identity with other snake venom metalloproteinases (SVMPs). BaltDC was devoid of proteolytic, hemorrhagic, defibrinating or coagulant activities, but it showed a specific inhibitory effect on platelet aggregation induced by ristocetin and epinephrine in PRP. IR analysis spectra strongly suggests that PO2−3PO32− groups, present in BaltDC, form hydrogen bonds with the PO−2PO2− groups present in the non-lipid portion of the membrane platelets.

 

Conclusions:

BaltDC may be of medical interest since it was able to inhibit platelet aggregation.

 

Keywords: Snake venom; Bothrops alternatus; DC protein; Platelet aggregation

 

Received: March 27., 2017.

Accepted: July 12, 2017.

 

Correspondence: juniacosta@iftm.edu.br

 

Authors’ contributions

MSM, BBS, DFCP, EHVD, CCNM and MRQ performed the experiments. ACAS and NOD performed the infrared experiments. AMS performed the N-terminal sequence. JOC participated in the analysis and discussion of the results and performed a critical revision of the work. FO was responsible for the design of the work and supervised all experiments. All authors contributed to writing of this manuscript and approved the final version.

 

Competing interests

The authors declare that they no competing interests.