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The Protein Journal

, Volume 37, Issue 4, pp 380–389 | Cite as

Biochemical Analyses of Proteins from Duttaphrynus melanostictus (Bufo melanostictus) Skin Secretion: Soluble Protein Retrieval from a Viscous Matrix by Ion-Exchange Batch Sample Preparation

  • Douglas O. C. Mariano
  • Marcela Di Giacomo Messias
  • José Pedro Prezotto-Neto
  • Patrick J. Spencer
  • Daniel C. Pimenta
Article
  • 160 Downloads

Abstract

A crucial step in scientific analysis can be sample preparation, and its importance increases in the same rate as the sensitivity of the following employed/desired analytical technique does. The need to analyze complex, viscous matrices is not new, and diverse approaches have been employed, with different success rates depending on the intended molecules. Solid-phase extraction, for example, has been successfully used in sample preparation for organic molecules and peptides. However, due to the usual methodological conditions, biologically active proteins are not successfully retrieved by this technique, resulting in a low rate of protein identification reported for the viscous amphibian skin secretion. Here we describe an ion-exchange batch processing sample preparation technique that allows viscous or adhesive materials (as some amphibian skin secretions) to be further processed by classical liquid chromatography approaches. According to our protocol, samples were allowed to equilibrate with a specific resin that was washed with appropriated buffers in order to yield the soluble protein fraction. In order to show the efficiency of our methodology, we have compared our results to classically prepared skin secretion, i.e., by means of filtration and centrifugation. After batch sample preparation, we were able to obtain reproductive resolved protein chromatographic profiles, as revealed by SDS-PAGE, and retrieve some biological activities, namely, hydrolases belonging to serine peptidase family. Not only that, but also the unbound fraction was rich in low molecular mass molecules, such as alkaloids and steroids, making this sample preparation technique also suitable for the enrichment of such molecules.

Keywords

Batch chromatography Ion-exchange chromatography Viscous sample Amphibian skin secretion Hydrolase 

Notes

Acknowledgements

We are thankful to Dr. Ivo Lebrun for the thoughtful considerations about the batch sample preparation and for providing the IEX media.

Funding

This work was funded by CAPES (DOCM, 969130 Grant), FINEP (Financiadora de Estudos e Projetos) (Grant Number 01.09.0278.04), FAPESP and CNPq. DCP is a CNPq fellow researcher (Conselho Nacional de Desenvolvimento Científico e Tecnológico) (Grant 303792/2016-7).

Compliance with Ethical Standards

Conflict of interest

Authors declare that they have no conflict of interest.

Ethical Approval

This article does not contain any studies with animals performed by any of the authors.

Supplementary material

10930_2018_9780_MOESM1_ESM.docx (930 kb)
Supplementary material 1 (DOCX 930 KB)

References

  1. 1.
    Duda TF, Vanhoye D, Nicolas P (2002) Roles of diversifying selection and coordinated evolution in the evolution of amphibian antimicrobial peptides. Mol Biol Evol 19:858–864CrossRefPubMedGoogle Scholar
  2. 2.
    Arnold DL, Jackson RW, Waterfield NR, Mansfield JW (2007) Evolution of microbial virulence: the benefits of stress. Trends Genet 23:293–300CrossRefPubMedGoogle Scholar
  3. 3.
    Brodie ED (2013) Convergent evolution: pick your poison carefully. Curr Biol 20:R152–R154CrossRefGoogle Scholar
  4. 4.
    Dowell NL, Giorgianni MW, Kassner VA, Selegue JE, Sanchez EE, Carroll SB (2016) The deep origin and recent loss of venom toxin genes in rattlesnakes. Curr Biol 26:2434–2445CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Toledo RC, Jared C (1995) Cutaneous granular glands and amphibian venoms. Comp Biochem Physiol 111:1–29CrossRefGoogle Scholar
  6. 6.
    Mendes VA, Barbaro KC, Sciani JM, Vassão RC, Pimenta DC, Jared C, Antoniazzi MM (2016) The cutaneous secretion of the casque-headed tree frog Corythomantis greeningi: biochemical characterization and some biological effects. Toxicon 122:133–141CrossRefPubMedGoogle Scholar
  7. 7.
    Sciani JM, Angeli CB, Antoniazzi MM, Jared C, Pimenta DC (2013) Differences and similarities among parotoid macrogland secretions in South American toads: a preliminary biochemical delineation. Sci World J 2013:1–9CrossRefGoogle Scholar
  8. 8.
    Mariano DOC, Yamaguchi LF, Jared C, Antoniazzi MM, Sciani JM, Kato MJ, Pimenta DC (2015) Pipa carvalhoi skin secretion profiling: absence of peptides and identification of kynurenic acid as the major constitutive component. Comp Biochem Physiol 167:1–6Google Scholar
  9. 9.
    Sousa-Filho LM, Freitas CD, Lobo MD, Monteiro-Moreira AC, Silva RO, Santana LA, Ribeiro RA, Souza MH, Ferreira GP, Pereira AC, Barbosa AL, Lima MS, Oliveira JS (2016) Biochemical profile, biological activities, and toxic effects of proteins in the Rhinella schneideri parotoid gland secretion. J Exp Zool A 325:511–523CrossRefGoogle Scholar
  10. 10.
    Rodríguez C, Rollins-Smith L, Ibáñez R, Durant-Archibold A, Gutiérrez M (2017) Toxins and pharmacologically active compounds from species of the family Bufonidae (Amphibia, Anura). J Ethnopharmacol 198:235–254CrossRefPubMedGoogle Scholar
  11. 11.
    Frost DR (2017) Amphibian species of the world: an online reference. Version 6.0. American Museum of Natural History, New York. http://research.amnh.org/herpetology/amphibia/index.html. Accessed 01 Feb 2017
  12. 12.
    Larsen EH, Ramlov H (2013) Role of cutaneous surface fluid in frog osmoregulation. Comp Biochem Physiol A 165:365–370CrossRefGoogle Scholar
  13. 13.
    Brodie ED (2009) Toxins and venoms. Curr Biol 19:R931–R935CrossRefPubMedGoogle Scholar
  14. 14.
    Conceição K, Bruni FM, Sciani JM, Konno K, Melo RL, Antoniazzi MM, Jared C, Lopes-Ferreira M, Pimenta DC (2009) Identification of bradykinin: related peptides from Phyllomedusa nordestina skin secretion using electrospray ionization tandem mass spectrometry after a single-step liquid chromatography. J Venom Anim Toxins Incl Trop Dis 15:633–652CrossRefGoogle Scholar
  15. 15.
    Vigerelli H, Sciani JM, Jared C, Antoniazzi MM, Caporale GM, da Silva Ade C, Pimenta DC (2014) Bufotenine is able to block rabies virus infection in BHK-21 cells. J Venom Anim Toxins Incl Trop Dis 20:1–8CrossRefGoogle Scholar
  16. 16.
    Sciani JM, de-Sá-Júnior PL, Ferreira AK, Pereira A, Antoniazzi MM, Jared C, Pimenta DC (2013) Cytotoxic and antiproliferative effects of crude amphibian skin secretions on breast tumor cells. Biomed Prev Nutr 3:10–18CrossRefGoogle Scholar
  17. 17.
    Sousa LQ, Machado KD, Oliveira SF, Araújo LD, Monção-Filho ED, Melo-Cavalcante AA, Vieira-Júnior GM, Ferreira PM (2017) Bufadienolides from amphibians: a promising source of anticancer prototypes for radical innovation, apoptosis triggering and Na+/K+-ATPase inhibition. Toxicon 127:63–76CrossRefPubMedGoogle Scholar
  18. 18.
    Vasu S, McGahon MK, Moffett RC, Curtis TM, Conlon JM, Abdel-Wahab YH, Flatt PR (2017) Esculentin-2CHa (1–30) and its analogues: stability and mechanisms of insulinotropic action. J Endocrinol 232:423–435CrossRefPubMedGoogle Scholar
  19. 19.
    Lipsky BA, Holroyd KJ, Zasloff M (2008) Topical versus systemic antimicrobial therapy for treating mildly infected diabetic foot ulcers: a randomized, controlled, double-blinded, multicenter trial of pexiganan cream. Clin Infect Dis 47:1537–1545CrossRefPubMedGoogle Scholar
  20. 20.
    König E, Wesse C, Murphy AC, Zhou M, Wang L, Chen T, Shaw C, Bininda-Emonds OR (2013) Molecular cloning of the trypsin inhibitor from the skin secretion of the Madagascan Tomato Frog, Dyscophus guineti (Microhylidae), and insights into its potential defensive role. Org Divers Evol 13:453–461CrossRefGoogle Scholar
  21. 21.
    Prates I, Antoniazzi MM, Sciani JM, Pimenta DC, Toledo LF, Haddad CF, Jared C (2012) Skin glands, poison and mimicry in dendrobatid and leptodactylid amphibians. J Morphol 273:279–290CrossRefPubMedGoogle Scholar
  22. 22.
    Weil AT, Davis W (1994) Bufo alvarius: a potent hallucinogen of animal origin. J Ethnopharmacol 41:1–8CrossRefPubMedGoogle Scholar
  23. 23.
    Evans CM, Brodie ED (1994) Adhesive strength of amphibian skin secretions. J Herpetol 28:499–502CrossRefGoogle Scholar
  24. 24.
    Anjolette FA, Leite FP, Bordon KC, Azzolini AEC, Pereira JC, Pereira-Crott LS, Arantes EC (2015) Biological characterization of compounds from Rhinella schneideri poison that act on the complement system. J Venom Anim Toxins Incl Trop Dis 21:25CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefPubMedGoogle Scholar
  26. 26.
    Prezotto-Neto JP, Kimura LF, Alves AF, Gutiérrez JM, Otero R, Suárez AM, Santoro ML, Barbaro KC (2016) Biochemical and biological characterization of Bothriechis schlegelii snake venoms from Colombia and Costa Rica. Exp Biol Med 241:2075–2085CrossRefGoogle Scholar
  27. 27.
    Cavalcante ID, Antoniazzi MM, Jared C, Pires OR Jr, Sciani JM, Pimenta DC (2017) Venomics analyses of the skin secretion of Dermatonotus muelleri: preliminary proteomic and metabolomic profiling. Toxicon 130:127–135CrossRefPubMedGoogle Scholar
  28. 28.
    Barker SA (2010) Matrix solid-phase dispersion. J Chromatogr 885:115–127CrossRefGoogle Scholar
  29. 29.
    Barker SA, Long AR, Short CR (1989) Isolation of drug residues from tissues by solid phase dispersion. J Chromatogr 475:353–361CrossRefPubMedGoogle Scholar
  30. 30.
    García-López M, Canosa P, Rodríguez I (2008) Trends and recent applications of matrix solid-phase dispersion. Anal Bioanal Chem 391:963–974CrossRefPubMedGoogle Scholar
  31. 31.
    Ramos L (2012) Critical overview of selected contemporary sample preparation techniques. J Chromatogr 122:84–98CrossRefGoogle Scholar
  32. 32.
    Pineda Guerra Y, BetancurEcheverri J, Pedroza-Díaz J, Delgado-Trejos E, Rothlisberger S (2016) Análisis proteómico del veneno de la abeja africanizada: comparación de métodos de extracción. Acta Biol Colomb 21:619–626CrossRefGoogle Scholar
  33. 33.
    Conceição K, Konno K, de Melo RL, Antoniazzi MM, Jared C, Sciani JM, Conceição IM, Prezoto BC, de Camargo AC, Pimenta DC (2007) Isolation and characterization of a novel bradykinin potentiating peptide (BPP) from the skin secretion of Phyllomedusa hypochondrialis. Peptides 28:515–523CrossRefPubMedGoogle Scholar
  34. 34.
    Rash LD, Morales RA, Vink S, Alewood PF (2011) De novo sequencing of peptides from the parotid secretion of the cane toad, Bufo marinus (Rhinella marina). Toxicon 57:208–216CrossRefPubMedGoogle Scholar
  35. 35.
    GE Healthcare (2010) Strategies for protein purification: handbook. GE Healthcare, UppsalaGoogle Scholar
  36. 36.
    Conceição K, Miriane Bruni F, Antoniazzi MM, Jared C, Camargo AC, Lopes-Ferreira M, Pimenta DC (2007) Major biological effects induced by the skin secretion of the tree frog Phyllomedusa hypochondrialis. Toxicon 49:1054–1062CrossRefPubMedGoogle Scholar
  37. 37.
    da Silva Libério M, Bastos IM, Júnior ORP, Fontes W, Santana JM, Castro MS (2014) The crude skin secretion of the pepper frog Leptodactylus labyrinthicus is rich in metallo and serine peptidases. PLoS ONE 9:e96893CrossRefGoogle Scholar
  38. 38.
    Rawlings ND, Barrett AJ (2013) Handbook of proteolytic enzymes. In: Introduction: serine peptidases and their clans. Academic Press, New York, pp 2491–2523Google Scholar
  39. 39.
    Kemparaju K, Girish KS (2006) Snake venom hyaluronidase: a therapeutic target. Cell Biochem Funct 24:7–12CrossRefPubMedGoogle Scholar
  40. 40.
    Kini RM, Koh CY (2016) Metalloproteases affecting blood coagulation, fibrinolysis and platelet aggregation from snake venoms: definition and nomenclature of interaction sites. Toxins 8:1–28CrossRefGoogle Scholar
  41. 41.
    Kawasaki H, Isaacson T, Iwamuro S, Conlon JM (2003) A protein with antimicrobial activity in the skin of Schlegel’s green tree frog Rhacophorus schlegelii (Rhacophoridae) identified as histone H2B. Biochem Biophys Res Commum 312:1082–1086CrossRefGoogle Scholar
  42. 42.
    Zhao Y, Jin Y, Lee WH, Zhang Y (2005) Isolation and preliminary characterization of a 22-kDa protein with trypsin inhibitory activity from toad Bufo andrewsi skin. Toxicon 46:277–281CrossRefPubMedGoogle Scholar
  43. 43.
    Bhattacharjee P, Giri B, Gomes A (2011) Apoptogenic activity and toxicity studies of a cytotoxic protein (BMP1) from the aqueous extract of common Indian toad (Bufo melanostictus Schneider) skin. Toxicon 57:225–236CrossRefPubMedGoogle Scholar
  44. 44.
    Zhang YX, Chen CW, Wang M, Wei SS, Guan H, Chi TT, Qi XZ, Hu WT (2011) Purification and characterization of albumin from frog skin of Duttaphrynus melanostictus. Protein J 30:464–470CrossRefPubMedGoogle Scholar
  45. 45.
    Zhao Y, Jin Y, Lee WH, Zhang Y (2006) Purification of a lysozyme from skin secretions of Bufo andrewsi. Comp Biochem Physiol C 142:46–52Google Scholar
  46. 46.
    Zhao Y, Jin Y, Wei SS, Lee WH, Zhang Y (2005) Purification and characterization of an irreversible serine protease inhibitor from skin secretions of Bufo andrewsi. Toxicon 46:635–640CrossRefPubMedGoogle Scholar
  47. 47.
    Zhang Y, Wang M, Wei S (2010) Isolation and characterization of a trypsin inhibitor from the skin secretions of Kaloula pulchrahainana. Toxicon 56:502–507CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Laboratório de Bioquímica e BiofísicaInstituto ButantanSão PauloBrazil
  2. 2.Centro de BiotecnologiaIPENSão PauloBrazil

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