PURIFICATION OF ANTIMICROBIAL PEPTIDES FROM AN EXTRACT OF THE SKIN OF XENOPUS-LAEVIS USING HEPARIN-AFFINITY HPLC - CHARACTERIZATION BY ION-SPRAY MASS-SPECTROMETRY

A simple scheme was developed for the rapid purification of antimicrobial peptides from the skin of Xenopus laevis. An extract of the frog skin was prepared using an acidic medium designed to maximize the solubilization of low-molecular-weight peptides. This extract was subjected to an enrichment procedure using C18 Sep Pak cartridges to yield a salt-free fraction, devoid of high-molecular-weight proteins. This fraction was in turn subjected to heparin affinity high-performance liquid chromatography on a Shodex AF-Pak column. All the antibacterial activity bound to the column and could be eluted using a linear gradient of increasing sodium chloride concentration. Antibacterial activity emerged from the column in fractions corresponding to a sodium chloride concentration of 0.45 M. Reversed-phase high-performance liquid chromatography resolved this material into a series of compounds which could be readily characterized using a combination of amino acid analysis and ion-spray mass spectrometry. Each peptide was found to be antimicrobial and each was positively identified as belonging to a family of amphipathic helix-forming peptides characterized by other investigators. Listed in their order of elution from the reversed-phase column the peptides were magainin 2, magainin 1, peptide-glycine-leucine amide, xenopsin precursor fragment, levitide precursor fragment, and a mixture of fragments derived from the caerulein precursor. These peptides owe their antimicrobial properties to a predeliction to forming amphipathic α-helical structures when associated with lipid membranes. The distribution of lysine residues within their sequences, while facilitating amphipathic helix formation, most likely also facilitates their binding to heparin. The basic peptides, xenopsin, levitide and metabolic fragments of magainin 2, were not bound by the heparin column. This suggested that the affinity of the antibacterial peptides for the affinity column is not solely a cation exchange phenomenon. A novel cystine-rich peptide 66 residues in length was also discovered in the nonretained fraction. Sequence analysis revealed that this peptide was structurally related to the neurotoxin/cytotoxin family previously found only in the venom of certain species of poisonous snakes. This study demonstrates how heparin-affinity chromatography can be utilized to purify basic amphipathic paptides and should prove useful in isolating similar antibacterial peptides from other sources such as the intestinal and pulmonary epithelial tissues of higher vertebrates. © 1994 Academic Press, Inc. All rights reserved.