SAXITOXIN

1. SAXITOXIN A Marine and Freshwater Toxin

2. Toxicity • Associated with “Red tides” • Origin: • Dinoflagellates • Gonyaulax tamarensis • Gonyaulax catenella • Cyanobacteria • Cylindrospermopsis raciborskii • Anabaena spiroides • Major transvectors are organisms feeding on producers • Molluscs, crabs, snails, etc.

3. Type and properties • Tetrahydropurine • Guanidinium • Heterocyclic • Hygroscopic • Water soluble • Thermally stable • MW of ~ 299g

4. STX and its derivatives STX has a total of about 20 different derivatives

5. Mechanism of Action • Absorbed through mucosae and excreted in urine • Blocks nerve signal transmission

6. Sodium Ion Channel • STX binds to receptor site extracellularly • Electrostatic attraction involving 7,8,9 guanidinium • Hydrogen-bonding involving C-12 hydroxyls • Located on α subunits • 4 homologous domains and 6 transmembrane segments • P-loop between S5 and S6 • S4 contains positive aa’s • EEDD outer and DEKA inner form selectivity filter and binding site

7. Nerve Propagation

8. Potassium Ion Channel • STX acts internally • Human ether-a-go-go-related gene • hERG • Associated with potassium ion current • Four α subunits and 6 transmembrane segments • Four domains surround central pore • STX binds 1:1 • Stabilizes the closed state

9. Voltage Activation Curves

10. hERG

11. Clinical Diagnoses • Symptoms appear with 2 hours of ingestion • Paralysis • Weakness • Headache • Nausea and vomiting • Death within 12 hours, depending on severity • Recovery in 2 to 7 days • No lasting side affects • Paralytic Shellfish Toxins (PSPs)

12. STX Studies • Classic form is Mouse Bioassay • Limit 0.4μg/g shellfish meat • Non-specific for STX • Enzyme Immunoassays • Developed by Johnson and Mulberry (1966) • Formaldehyde condensation • Antitoxin produced in rabbits • Antiserum + STX used in hemagluttination and bentonite flocculation tests • Toxin precipitated • Specific for STX, but only qualitatively

13. More STX Studies • Fluorimetric determination • Acidic extraction and alkaline oxidation • Spectrophotometric • Limit .004μg/g meat • Could not detect different PSPs • Crown ethers • Anthracyl group serves as a fluorescent probe • STX binds through C-8 and C-12

14. General Structure

15. STX Docked In Crown

16. Last But Not Least • HPLC • Bonded cyano column • Alkiline oxidation using periodic acid and sodium phosphate buffer • Six individual PSPs identified and quantified • Limit 0.2fg/g

17. Natural Detection • Saxiphilin (SAX) • Compound isolated from N.A. bullfrog • Rana catesbeiana • Soluble protein • Detected using tritiated-STX (H3-STX) • Specific and high affinity for STXs • Derivatives of STXs and SAXs used as radioligands • Measure sodium density channels • Does not bind STX competitor TTX

18. Saxiphilin (cont.) • Structure • N-lobe and C-lobe • Binding site for STX on C-lobe • Cloning and sequencing cDNA • ~90-92 kDa protein • Related to transferrin family • Purpose and action still unknown • Maybe a species-specific detoxification mechanism • STX has high affinity for SAX

19. Conclusion • STXs are dangerous marine and freshwater toxins • Biosynthesis still unknown • Valuable for medical research • Nervous system disorder • Implicated for use in chemical warfare • No cure known

20. References • Edwards, Neil. "Saxitoxin...from food poisoning to chemical warfare...." The Chemical Laboratories. 18 Aug. 1998. University of Sussex at Brighton. 14 Nov. 2005 http://www.bris.ac.uk/Depts/Chemistry/MOTM/stx/saxi1.htm. • "Environmental Health Criteria 37: Aquatic (Marine and Freshwater) Biotoxins." International Programme on Chemical Safety. 1984. World Health Organization. 14 Nov. 2005 <http://www.inchem.org/documents/ehc/ehc/ehc37.htm>. • Fleming, Lora E. "Paralytic Shellfish Poisoning." NIEGS Marine and Freshwater Biomedical Sciences. 14 Nov. 2005 <http://www.whoi.edu/science/B/redtide/illness/psp.html>. • Gawley, R.E., Pinet, S., Cardona, C.M., Datta, P.K., Ren, T., Guida, W.C., Nydick, J., and Leblanc, R.M. J. Am. Chem. Soc., 124, 45, 13448 - 13453, 2002, 10.1021/ja027507p. • Goldin, Alan L. "Mechanisms of Sodium Channel Inactivation." Current Opinion in Neurolbiology 13, 284-290 (2003). 2 Dec 2005 <http://www.biol.sc.edu/~vogt/courses/neuro/pdf/Na-channel-inactivation-Goldin-CurrOpNeuro-2003.pdf>. • Jixin Wang, Joseph J. Salata, and Paul B. Bennett. “Saxitoxin Is a Gating Modifier of hERG K+ Channels.” J. Gen. Physiol., 121: 583-598; published online before print as 10.1085/jgp.200308812. • Krishnan, Gomathi, Maria A. Morabito, and Edward Moczydlowski*. "Expression and characterization of Flag-epitope- and hexahistidine-tagged derivatives of saxiphilin for use in detection and assay of saxitoxin." Toxicon 39, 291-301 (2001). 27 Nov 2005 <http://people.clarkson.edu/~emoczydl/niallpaper1.pdf>. • Li, Yi , and Edward Moczydlowski. "Purification and Partial Sequencing of Saxiphilin, a Saxitoxin-binding Protein from the Bullfrog, Reveals Homology to Transferrin*." The Journal of Biological Chemistry 266, 23, 15481-15487 (1991). 27 Nov 2005 <http://people.clarkson.edu/~emoczydl/niallpaper3.pdf>. • Llewellyn, Lyndon E., and Edward G. Moczydlowski. "Characterization of Saxitoxin Binding to Saxiphilin, a Relative of the Transferring Family That Displays pH-Dependent Ligand Binding." Biochemistry 33, 12312-12322 (1994). 25 Nov 2005 <http://pubs.acs.org/cgi-bin/abstract.cgi/bichaw/1994/33/i40/f-pdf/f_bi00206a039.pdf?sessid=6006l3>. • Penzotti, Jennifer L., Fozzard, Harry A., Lipkind, Gregory M., Dudley, Samuel C., Jr.”Differences in Saxitoxin and Tetrodotoxin Binding Revealed by Mutagenesis of the Na+ Channel Outer Vestibule.” Biophys. J., 1998, 75: 2647-2657. • “Theoretical Study of the Interaction between the Guanidinium Cation and Chloride and Sulfate Anions.” Rozas, I. and Kruger, P.E. J. Chem. Theory Comput., 1, 5, 1055 - 1062, 2005, 10.1021/ct050009x. • "Various Shellfish-Associated Toxins." Foodborne Pathogenic Microorganisms and Natural Toxins Handbook. Jan. 1992. U.S. Food and Drug Administration. 14 Nov. 2005 <http://vm.cfsan.fda.gov/~mow/chap37.html>.