Identification and Analysis of Bioactive Peptides in Amphibian Skin Secretions
Skin secretions from anurans (frogs and toads), particularly those species belonging to the Hylidae and Ranidae families, are a rich source of biologically active peptides. Cytolytic peptides with broad-spectrum antimicrobial activities and highly variable amino acid sequences are often released into these secretions in high concentrations. Identification and characterization of these components can prove to be valuable in species identification, elucidation of evolutionary histories and phylogenetic relationships between species, and may lead to development of agents with potential for therapeutic application. This chapter describes the use of norepinephrine (injection or immersion) to stimulate peptide release in a procedure that does not appear to cause distress to the animals. The peptide components in the secretions are separated by reversed-phase HPLC on octadecylsilyl silica (C18) columns under standard conditions after partial purification on Sep-Pak cartridges. Individual peptides are identified by determination of their molecular masses by MALDI-TOF mass spectrometry and from their retention times. The use of mixtures of synthetic peptides of appropriate molecular mass as calibration standards enables mass determination to a high degree of precision.
Key wordsFrog skin secretions antimicrobial peptide reversed-phase HPLC MALDI-TOF mass spectrometry
- 6.Vanhoye, D., Bruston, F., Nicolas, P. and Amiche, M. (2003) Antimicrobial peptides from hylid and ranin frogs originated from a 150-million-year-old ancestral precursor with a conserved signal peptide but a hypermutable antimicrobial domain. Eur. J. Biochem. 270, 2068–2081.PubMedCrossRefGoogle Scholar
- 10.Frost, D.R. (2008) Amphibian species of the world: an online reference. Version 5.2. Electronic database accessible at http://research.amnh.org/herpetology/amphibia/index.php American Museum of Natural History, New York.
- 13.Goraya, J., Wang, Y., Li, Z., O‘Flaherty, M., Knoop, F.C., Platz, J.E., et al. (2000) Peptides with antimicrobial activity from four different families isolated from the skins of the North American frogs Rana luteiventris, Rana berlandieri and Rana pipiens. Eur. J. Biochem. 267, 894–900.PubMedCrossRefGoogle Scholar