Resonant mirror biosensor detection method based on yessotoxin-phosphodiesterase interactions.
Yessotoxin (YTX) is a generic name for a group of lipophilic compounds recently discovered and chemically characterized. Association measurements were done in a resonant mirror biosensor. The instrument detects changes in the refractive index and/or thickness occurring within a few hundred nanometers form the sensor surface where a molecule is attached. We used aminosilane surfaces where phosphodiesterase 3',5'-cyclic-nucleotide-specific from bovine brain (PDEs) was immobilized. Over this immobilized ligand different amounts of YTX were added and typical association curve profiles were observed. These association curves fit a pseudo-first-order kinetic equation where the apparent association rate constant (k(on)) can be calculated. The value of this constant increases with YTX concentration. From the representation of k(on) versus YTX concentration we obtained the association rate constant (k(ass)) 248+/-40 M(-1)s(-1) and the dissociation rate constant (k(diss)) 9.36 x 10(-4)+/-1.72 x 10(-4)s(-1). From these values the kinetic equilibrium dissociation constant (K(D)) for YTX-PDEs association can be calculated. The value of this last constant is 3.74 x 10(-6)+/-8.25 x 10(-8)M YTX. The PDE-YTX association was used as a method suitable for determination of the toxin concentration in a shellfish sample. The assay had sufficient sensitivity and can be used on simple shellfish extracts.
Pazos
María-José
MJ
Departamento de Farmacología, Facultad de Veterinaria, USC, 27002 Lugo, Spain.
Alfonso
Amparo
A
Vieytes
Mercedes R
MR
Yasumoto
Takeshi
T
Vieites
Juan M
JM
Botana
Luis M
LM
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism*; D000818:Animals; D015374:Biosensing Techniques; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D004347:Drug Interactions; D004988:Ethers, Cyclic / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008978:Mollusk Venoms / Q000032:analysis*; D035964:Oxocins / Q000737:chemistry / Q000378:metabolism*; D012031:Refractometry; D012758:Shellfish; D012821:Silanes / Q000378:metabolism
D004988:Ethers, Cyclic; D008024:Ligands; D008978:Mollusk Venoms; D035964:Oxocins; D012821:Silanes; D015105:3',5'-Cyclic-AMP Phosphodiesterases; C066632:yessotoxin