A half century ago in 1957, B.C. Abbot and D. Ballantine described the partial purification and characterization of a toxin from G. veneficum. To paraphrase their findings, “…The toxin molecule must be large, as it cannot penetrate a dialysis membrane; it is soluble in water and the lower alcohols, but insoluble in ether and chloroform. It is unstable in acids, … though in neutral solution is more or less thermostable. …With regard to mode of action it depolarizes nerve and muscle membranes. …This depolarization probably occurs by interference with the sodium exchange mechanism, allowing rapid entry of sodium into the cells." This species has subsequently been renamed Karlodinium veneficum and we describe here for the first time the absolute structure (KmTX2) of this toxin. We have named these toxins, karlotoxins, with a molecular formula of C67H121ClO24 for KmTX2. Although the individual toxins produce a single C18 HPLC signal, MS revealed congeners co-eluting within each HPLC signal. These congeners differ from each other by a single hydroxylation and can be separated under normal phase NH2 chromatography. Our hypothesis is that Karlodinium was responsible for the large fish-kills previously associated with Pfiesteria. This hypothesis is supported by the isolation of karlotoxin at fish kill events from around the world. KmTX2 is lethal to fish at concentrations measured during fish kills, while sublethal doses damage gill epithelia. Cellular toxicity occurs through permeabilization of plasma membranes, resulting in osmotic lysis. Membrane sterol composition is an important determinant of KmTx2 activity and appears to play a role in the immunity of K. veneficum against its own toxins. The structural assignment of this molecule was facilitated by 13C enrichment through growth on NaH13CO3 in culture. This enriched material combined with the use of a cyro-dual probe allowed for the acquisition of INADEQUATE results with just 1-2 mgs of material. In addition the quality of the inverse experiments was significantly enhanced for enriched material allowing the assignment of the gross structure for the toxin. The assignment of absolute configuration has been recently completed and will be presented here for the first time.
Presenter:
Mark T. Hamann, Ph.D.
University of Mississippi School of Pharmacy and Department of Chemistry & Biochemistry
Friday, June 26, 2009
10:30 a.m.
Pacific Ocean Science & Technology (POST) 723
The Pacific Research Center for Marine Biomedicine (PRCMB) is a newly established center at the University of Hawaii dedicated to trans-disciplinary research designed to gain new knowledge about the profound impacts of the ocean on human health. The Center is funded by the National Science Foundation and the National Institute of Environmental Health Sciences.
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