Preparation and characterization of a biologically active spin-labeled sea anemone toxin

Stephen A. Monks, Raymond S. Norton, Cyril C. Curtain, Lawrence J. Berliner

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A derivative of the polypeptide cardiostimulant anthopleurin-B(AP-B) labeled with the spin label 1-oxyl 2,2,6,6-tetramethyl-4- piperidinyloxycarbonyl azide has been prepared and characterized. The product was found by mass spectrometry to be labeled at a single site, which amino acid sequencing showed to be the N-terminus. It also retained positive inotropic activity when assayed on isolated guinea pig atria. The spin- labeled (SL) product was found to exist in two distinct conformations by reversed-phase HPLC and in at least two conformations by electron spin resonance spectroscopy (ESR) over the pH range 2-9. The ESR data also show evidence for multimetric states of SL-AP-B over the pH range 2-9, with maximum aggregation at pH 4.5-5, and a slow disaggregation when the pH is adjusted to 8-9. The presence of multiple conformers of SL-AP-B and its tendency to aggregate render it unsuitable for high-resolution NMR structural studies of the isolated ligand, but the retention of activity may make it useful for studies of the sodium-channel-bound form of the molecule.

Original languageEnglish
Pages (from-to)427-434
Number of pages8
JournalJournal of Protein Chemistry
Issue number5
Publication statusPublished - 1996
Externally publishedYes


  • Aggregation
  • Anthopleurin
  • ESR
  • Multiple conformers
  • Spin label

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