Quantification of yessotoxin using the fluorescence polarization technique and study of the adequate extraction procedure.
Yessotoxin (YTX) is a polycyclic ether toxin produced by phytoplanktonic microalgae from the group of dinoflagellates. It has been shown that YTX increases the 3',5'-cyclic nucleotide phosphodiesterases (PDEs) activity and that there is a binding between these proteins and the toxin. Fluorescence polarization (FP) is a spectroscopic technique that can be used to study the interactions between molecules. It is based on exciting a fluorescent molecule with plane-polarized light and measuring the polarization degree of the emitted light. In this study, the FP is applied to the study of the interaction between YTX and phosphodiesterases I and II (PDE I and II). The phosphodiesterases are labeled with a reactive succinimidyl esther of carboxyfluorescein, and the FP of the protein-dye conjugate is measured when the YTX concentration in the medium increases. The results show that in both cases the fluorescence polarization of the conjugates decreases when they bind to YTX. For the PDE I, it is possible to draw a Gaussian curve or a straight line that relates the two variables (FP and YTX concentration). The concentration of this toxin in a spiked mussel extract (which contains the conjugate) can be quantified measuring its FP and using the equations of those lines. Different extraction methods are tried in this study, and those that can be used to obtain an appropriate mussel extract to be quantified with this technique are determined.
Alfonso
Carmen
C
Departamento de Farmacología, Facultad de Veterinaria, USC, 27002 Lugo, Spain.
Alfonso
Amparo
A
Vieytes
Mercedes R
MR
Yasumoto
Takeshi
T
Botana
Luis M
LM
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism*; D000818:Animals; D049872:Bivalvia / Q000737:chemistry; D005591:Chemical Fractionation / Q000379:methods; D004988:Ethers, Cyclic / Q000032:analysis* / Q000378:metabolism; D005454:Fluorescence Polarization / Q000379:methods; D008978:Mollusk Venoms / Q000032:analysis; D035964:Oxocins / Q000032:analysis* / Q000378:metabolism
D004988:Ethers, Cyclic; D008978:Mollusk Venoms; D035964:Oxocins; D015105:3',5'-Cyclic-AMP Phosphodiesterases; C066632:yessotoxin