Quenching of bathocuproine disulfonate fluorescence by Cu(I) as a basis for copper quantification.
In this paper we report the up to now ignored fluorescence properties of the specific Cu(I)-chelator bathocuproine disulfonate and their application in assays of total copper and Cu(I). The method is based on the linear quenching of the bathocuproine disulfonate emission at 770 nm (lambda(ex)580 nm) by increasing concentrations of Cu(I), at pH 7.5. Copper concentrations as low as 0.1 microM can be determined. Other metal ions (iron, manganese, zinc, cadmium, cobalt, nickel) do not interfere. The procedure for total copper determination in proteins includes HCl treatment to release the copper, neutralization to pH 7.5 in the presence of citrate to stabilize the copper, and reduction of the copper to Cu(I) by ascorbate in the presence of the chelator. This assay gave results coincident with the analysis by atomic absorption spectroscopy in two selected proteins. In addition, conditions are described (omitting HCl treatment and reduction by ascorbate) for direct measurement of Cu(I) in native proteins, as illustrated for the Escherichia coli NADH dehydrogenase-2. Data show that the fluorometric assays described in this paper are simple and convenient procedures for total copper and direct Cu(I) quantification in determined biological samples.
Departamento Bioquímica de la Nutrición, Instituto Superior de Investigaciones Biológicas, Consejo Nacional de Investigaciones Científicas y Tecnológicas, Universidad Nacional de Tucumán, San Miguel de Tucumán, Argentina.
Viviana A
VA
Rapisarda
Sabrina I
SI
Volentini
Ricardo N
RN
Farías
Eddy M
EM
Massa
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002614:Chelating Agents / Q000737:chemistry; D003300:Copper / Q000032:analysis*; D004912:Erythrocytes / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology; D005453:Fluorescence; D006851:Hydrochloric Acid / Q000378:metabolism; D007202:Indicators and Reagents; D009245:NADH Dehydrogenase / Q000737:chemistry / Q000378:metabolism; D009942:Organometallic Compounds / Q000737:chemistry*; D010084:Oxidation-Reduction; D010618:Phenanthrolines / Q000737:chemistry* / Q000378:metabolism; D013054:Spectrophotometry, Atomic; D013482:Superoxide Dismutase / Q000378:metabolism
D002614:Chelating Agents; D007202:Indicators and Reagents; D009942:Organometallic Compounds; D010618:Phenanthrolines; C048402:copper(I)-thiolate; C028559:bathocuproine sulfonate; D003300:Copper; D013482:Superoxide Dismutase; C088908:NADH dehydrogenase II; D009245:NADH Dehydrogenase; D006851:Hydrochloric Acid