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 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.
Monks
S A
SA
NMR Laboratory, Biomolecular Research Institute, Parkville, Australia.
Norton
R S
RS
Curtain
C C
CC
Berliner
L J
LJ
D000818:Animals; D001386:Azides; D002851:Chromatography, High Pressure Liquid; D004578:Electron Spin Resonance Spectroscopy; D006863:Hydrogen-Ion Concentration; D036341:Intercellular Signaling Peptides and Proteins; D007536:Isomerism; D008387:Marine Toxins / Q000737:chemistry* / Q000302:isolation & purification; D010455:Peptides / Q000737:chemistry* / Q000302:isolation & purification; D012615:Sea Anemones / Q000737:chemistry*; D013113:Spin Labels
D001386:Azides; D036341:Intercellular Signaling Peptides and Proteins; D008387:Marine Toxins; D010455:Peptides; D013113:Spin Labels; C026132:anthopleurin B