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 alpha-helical structures when associated with lipid membranes.(ABSTRACT TRUNCATED AT 250 WORDS)
James
S
S
Endocrine Laboratory, Royal Victoria Hospital, Montreal, Quebec, Canada.
Gibbs
B F
BF
Toney
K
K
Bennett
H P
HP
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000900:Anti-Bacterial Agents / Q000302:isolation & purification; D000890:Anti-Infective Agents / Q000302:isolation & purification*; D023181:Antimicrobial Cationic Peptides; D002846:Chromatography, Affinity / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005260:Female; D006493:Heparin; D054806:Magainins; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D010455:Peptides / Q000008:administration & dosage / Q000302:isolation & purification*; D017421:Sequence Analysis; D012867:Skin / Q000737:chemistry*; D029867:Xenopus Proteins; D014982:Xenopus laevis
D000596:Amino Acids; D000900:Anti-Bacterial Agents; D000890:Anti-Infective Agents; D023181:Antimicrobial Cationic Peptides; D054806:Magainins; D010455:Peptides; D029867:Xenopus Proteins; C053301:magainin 2 peptide, Xenopus; C053300:magainin 1 peptide, Xenopus; D006493:Heparin