A fluorescent microplate assay for microcystin-LR.
A fluorescent enzyme inhibition assay for microcystin-LR was developed using a new fluorescent substrate of protein phosphatases 1 (PP1) and 2A (PP2A), 6,8-difluoro-4-methylumbelliferyl phosphate. The PP1 and PP2A inhibition assay for microcystin-LR was performed in a microtiter plate and the fluorescence yielded by the enzymatic hydrolysis of the substrate was quantified in a fluorescence plate reader. The concentration of microcystin-LR causing 50% inhibition of PP1 and PP2A activity (IC50) was 0.01 nM for PP1 and 0.08 nM for PP2A. The measurable range of microcystin-LR was 800 to 0.08 pg/well for both enzymes. The described assay is fast and very sensitive for the detection of microcystin-LR. Furthermore, this assay can be successfully applied to the study of toxins that inhibit PP1 or PP2A.
Fontal
O I
OI
Facultad de Veterinaria, Universidad de Santiago de Compostela, Lugo, 27002, Spain.
Vieytes
M R
MR
Baptista de Sousa
J M
JM
Louzao
M C
MC
Botana
L M
LM
D000458:Cyanobacteria / Q000737:chemistry; D005456:Fluorescent Dyes; D006923:Hymecromone / Q000031:analogs & derivatives; D007700:Kinetics; D008387:Marine Toxins; D052998:Microcystins; D010456:Peptides, Cyclic / Q000032:analysis*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods* / Q000706:statistics & numerical data; D013379:Substrate Specificity
C119285:6,8-difluoro-4-methylumbelliferyl phosphate; D005456:Fluorescent Dyes; D008387:Marine Toxins; D052998:Microcystins; D010456:Peptides, Cyclic; D006923:Hymecromone; D010749:Phosphoprotein Phosphatases; C057862:cyanoginosin LR