title: Assessment of protein phosphatase in a re-usable rapid assay format in detecting microcystins and okadaic acid as a precursor to biosensor development.
abstract: The feasibility of developing an immobilised protein phosphatase (PP) biosensor was tested by immobilising PP onto CNBr-activated Sepharose beads placed in Millipore microfilter plate wells. Under optimised immobilised enzyme assay conditions, okadaic acid (OA) and microcystin LR (MC-LR) inhibited Upstate Biotechnology PP (PP-2A), with IC50 values of 12.5 and 11nM respectively. Similarly, immobilised recombinant PP type 1 (rec PP-1) was inhibited by MC-LR and OA, with IC50 values of 150 and >1000nM respectively. The IC50 values for free PP-2A against OA and MC-LR were 2.5 and 3.5nM, and 0.7nM and 200nM for rec PP-1 against the same substrates respectively. For free and immobilised Neptunea arthritic PP (PP-2Ana) against OA the IC50 values were 0.45 and >1000nM respectively. Of the three immobilised enzyme systems, PP-2A showed greatest sensitivity to OA and MC-LR followed by rec PP-1 and PP-2Ana. In assessments for re-usability (determined by removal of > or =70% OA or MC-LR inhibition of PP-2A by washing), <50% of the original activity remained after 20 washings. Including 1M NaCl in the wash buffer did not increase enzyme activity with wash frequency, but rather "salted in" the inhibitor. The LoD of immobilised PP-2A to MC-LR meets the WHO guideline of 1microgl(-1) for drinking water, and the sensitivity to OA (3.5microgl(-1)) would allow detection of DSP during the peak of some phytoplankton blooms.
authors.affiliation: Agricultural and Life Sciences Division, PO Box 84, Lincoln University, Canterbury, New Zealand.
authors.forename: L L
authors.initials: LL
authors.lastname: Allum
authors.forename: D O
authors.initials: DO
authors.lastname: Mountfort
authors.forename: R
authors.initials: R
authors.lastname: Gooneratne
authors.forename: N
authors.initials: N
authors.lastname: Pasco
authors.forename: G
authors.initials: G
authors.lastname: Goussain
authors.forename: E A H
authors.initials: EA
authors.lastname: Hall
mesh_terms: D000818:Animals; D015374:Biosensing Techniques / Q000379:methods* / Q000592:standards; D004800:Enzymes, Immobilized / Q000737:chemistry*; D049851:Gastropoda / Q000737:chemistry*; D052998:Microcystins / Q000032:analysis*; D019319:Okadaic Acid / Q000032:analysis*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry*
chemical_list: D004800:Enzymes, Immobilized; D052998:Microcystins; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases