Colorimetric and electrochemical phosphodiesterase inhibition assays for yessotoxin detection: development and comparison with LC-MS/MS.
This work describes the development and applicability of two functional assays for the detection of yessotoxin (YTX), a polycyclic ether marine toxin produced by dinoflagellates. The assays are based on the interaction between this toxin and the phosphodiesterase (PDE) enzyme and the subsequent measurement of the enzyme activity by colorimetric and electrochemical methods. Firstly, several enzyme substrates were tested in order to select those able to be detected by colorimetry or electrochemistry after enzymatic hydrolysis. The substrates that provided the highest absorbance values and density currents were p-nitrophenyl phenylphosphonate and alpha-naphthyl phosphate, respectively. After optimisation of the experimental parameters, limits of detection of 0.8 and 0.6 microM were attained by colorimetry and electrochemistry, respectively. An inhibitory effect of YTX on the PDE activity was observed. The assays have been applied to the analysis of YTX production by Protoceratium reticulatum cultures, and results were compared with liquid chromatography-tandem mass spectrometry analysis.
IRTA, Ctra. Poble Nou, km. 5.5, 43540 Sant Carles de la Ràpita, Spain. monica.campas@irta.cat
Mònica
M
Campàs
Pablo
P
de la Iglesia
Margarita
M
Fernández-Tejedor
Jorge
J
Diogène
D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods; D003124:Colorimetry / Q000379:methods*; D017839:Crotalus / Q000378:metabolism; D004141:Dinoflagellida / Q000737:chemistry*; D004563:Electrochemistry; D057230:Limit of Detection; D008978:Mollusk Venoms; D035964:Oxocins / Q000032:analysis*; D010726:Phosphodiesterase Inhibitors / Q000032:analysis*; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry*; D011485:Protein Binding; D053719:Tandem Mass Spectrometry / Q000379:methods
D008978:Mollusk Venoms; D035964:Oxocins; D010726:Phosphodiesterase Inhibitors; D010727:Phosphoric Diester Hydrolases; C066632:yessotoxin