title: Palytoxin detection and quantification using the fluorescence polarization technique.
abstract: Palytoxin (PLT) is a highly toxic nonpeptidic marine natural product, with a complex chemical structure. Its mechanism of action targets Na,K-ATPase. Fluorescence polarization (FP) is a spectroscopic technique that can be used to determine molecular interactions. It is based on exciting a fluorescent molecule with plane-polarized light and measuring the polarization degree of the emitted light. In this study, FP was used to develop a detection method based on the interaction between the Na,K-ATPase and the PLT. The Na,K-ATPase was labeled with a reactive succinimidyl esther of carboxyfluorescein, and the FP of protein-dye conjugate was measured when the amount of PLT in the medium was modified. The assay protocol was first developed using ouabain as a binding molecule. The final result was a straight line that correlates FP units and PLT concentration. Within this line the PLT equivalents in a natural sample can be quantified. A selective cleaning procedure to mussel samples and dinoflagellates cultures was also developed to avoid the matrix effect. The LOQ (limit of quantification) of the method is 10nM and the LOD (limit of detection) is 2 nM. This new PLT detection method is easier, faster, and more reliable than the other methods described to date.
authors.affiliation: Department of Pharmacology, Veterinary School, 27002 Lugo, Spain.
authors.forename: A
authors.initials: A
authors.lastname: Alfonso
authors.forename: A
authors.initials: A
authors.lastname: Fernández-Araujo
authors.forename: C
authors.initials: C
authors.lastname: Alfonso
authors.forename: B
authors.initials: B
authors.lastname: Caramés
authors.forename: A
authors.initials: A
authors.lastname: Tobio
authors.forename: M C
authors.initials: MC
authors.lastname: Louzao
authors.forename: M R
authors.initials: MR
authors.lastname: Vieytes
authors.forename: L M
authors.initials: LM
authors.lastname: Botana
mesh_terms: D000178:Acrylamides / Q000032:analysis*; D000818:Animals; D049872:Bivalvia / Q000737:chemistry; D003064:Cnidarian Venoms; D004141:Dinoflagellida / Q000737:chemistry; D005454:Fluorescence Polarization / Q000379:methods*; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D010042:Ouabain / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013696:Temperature; D014020:Tissue Extracts / Q000737:chemistry; D000095702:Polyether Toxins
chemical_list: D000178:Acrylamides; D003064:Cnidarian Venoms; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase; D014020:Tissue Extracts; C010272:palytoxin; D000095702:Polyether Toxins