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-piperidinylox ycarbonyl azide has been prepared and characterized, The product was f ound 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 spi n-labeled (SL) product was found to exist in two distinct conformation s 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 rang e 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 o f SL-AP-B and its tendency to aggregate render it unsuitable for high- resolution NMR structural studies of the isolated ligand, but the rete ntion of activity may make it useful for studies of the sodium-channel -bound form of the molecule.