title: A fluorescence-based sensing system for the environmental monitoring of nickel using the nickel binding protein from Escherichia coli.
abstract: A sensing system for nickel based on the nickel binding protein (NBP) from Escherichia coli is shown to be feasible. The versatility of NBP was demonstrated by its use in three different assay formats. When the NBP binds nickel, it undergoes a conformational change that can be used as the basis for an optical sensing system for nickel. The NBP gene was overexpressed in E. coli and the protein purified in a single step using perfusion anion-exchange chromatography. A unique cysteine residue at position 15 in the NBP was labeled with the fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC). In a spectrofluorimetric assay, there was a maximum of 65% quenching of the fluorescence signal produced by NBP-MDCC in the presence of nickel. A response curve for nickel using NBP-MDCC revealed a detection limit of 8 x 10(-8) mol L(-1). NBP-MDCC was also used to develop assays in microtiter plate and fiber optic bundle formats. Detection limits for nickel using these formats were also in the submicromolar range. Selectivity studies conducted with other divalent metals, including copper, cobalt, iron, cadmium, and manganese, showed that fluorescence quenching for cobalt was similar in magnitude but with a detection limit more than 10-fold higher than for nickel. The quenching responses were lower for the other metals, with detection limits at least 10 to 100 times higher than for nickel. These results suggest that fluorescently labeled NBP is potentially useful in the development of a sensing system for nickel.
authors.affiliation: Department of Chemistry, University of Kentucky, Lexington 40506-0055, USA.
authors.forename: Lyndon L E
authors.initials: LL
authors.lastname: Salins
authors.forename: Elizabeth S
authors.initials: ES
authors.lastname: Goldsmith
authors.forename: C Mark
authors.initials: CM
authors.lastname: Ensor
authors.forename: Sylvia
authors.initials: S
authors.lastname: Daunert
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis* / Q000378:metabolism; D002352:Carrier Proteins / Q000032:analysis* / Q000378:metabolism; D003374:Coumarins; D004264:DNA Topoisomerases, Type I / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004784:Environmental Monitoring; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005456:Fluorescent Dyes; D009532:Nickel / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D003374:Coumarins; D005456:Fluorescent Dyes; C068025:nikA protein, Plasmid R64; C088410:N-(2-(1-maleimidyl)ethyl)-7-(diethylamino)coumarin-3-carboxamide; D009532:Nickel; D004264:DNA Topoisomerases, Type I