A bienzyme electrochemical probe for flow injection analysis of okadaic acid based on protein phosphatase-2A inhibition: an optimization study.
A bienzyme electrochemical probe has been assembled and used to monitor the inhibition of the enzyme protein phosphatase-2A (PP2A) by okadaic acid (OA), taking advantage of the particular characteristics of a biochemical pathway in which PP2A is involved. This enzyme has significant activity toward glycogen phosphorylase a (PHOS a), which in turn catalyzes the conversion of glycogen to glucose-1-phosphate (G-1-P). In addition, PP2A is strongly inhibited by OA and its derivatives. Due to this combination of properties, PP2A was employed to develop an assay system involving a preliminary phase of off-line enzymatic incubations (OA/PP2A, PP2A/PHOS a, PHOS a/glycogen+phosphate). This off-line step was followed by the electrochemical detection of H2O2, which is the final product of two sequential enzymatic reactions: G-1-P with alkaline phosphatase (AP) producing glucose, then glucose with glucose oxidase (GOD) producing hydrogen peroxide. These two enzymes were coimmobilized on a nylon net membrane that was placed over an H2O2 platinum probe inserted into a flow injection analysis (FIA) system. During a first phase of the study, all analytical parameters were optimized. During a subsequent phase, the inhibition of PP2A enzyme by OA was evaluated. The calibration of the system shows a working range for detection of OA between 30 and 250 pg ml(-1). The total analysis time is the sum of 50 min for the off-line enzymatic incubations and 4 min for the biosensor response.
Volpe
G
G
Dipartimento di Scienze e Tecnologie Chimiche, Università di Roma Tor Vergata, 00133 Roma, Italy. giulia.volpa@uniroma2.it
Cotroneo
E
E
Moscone
D
D
Croci
L
L
Cozzi
L
L
Ciccaglioni
G
G
Palleschi
G
G
D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D004563:Electrochemistry; D004566:Electrodes; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000494:pharmacology*; D004795:Enzyme Stability; D017022:Flow Injection Analysis / Q000295:instrumentation / Q000379:methods*; D006003:Glycogen / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D019319:Okadaic Acid / Q000032:analysis* / Q000494:pharmacology*; D009994:Osmolar Concentration; D010710:Phosphates / Q000737:chemistry; D054648:Protein Phosphatase 2 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D012680:Sensitivity and Specificity
D004791:Enzyme Inhibitors; D010710:Phosphates; D019319:Okadaic Acid; D006003:Glycogen; D054648:Protein Phosphatase 2