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

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 thepH range 2–9. The ESR data also show evidence for multimetric states of SL-AP-B over thepH range 2–9, with maximum aggregation at pH 4.5–5, and a slow disaggregation when thepH 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.Abbreviations AP-A anthopleurin-A - AP-B anthopleurin-B - ATX Ia toxin Ia fromAnemonia sulcata - Sh I neurotoxin I fromStichodactyla helianthus - TFA trifluoroacetic acid - SL-AP-B AP-B labeled at the N-terminus with the spin label 1-oxyl 2,2,6,6-tetramethyl-4-piperidinyloxycarbonyl azide