DETERMINATION OF PICOMOLAR CONCENTRATIONS OF METAL LENS USING FLUORESCENCE ANISOTROPY - BIOSENSING WITH A REAGENTLESS ENZYME TRANSDUCER

Because of their high affinity and selectivity, metalloproteins can be used as transducers in novel sensors, i.e., biosensors, for the deter mination of trace levels of metal ions in solution. Here, we exploit c arbonic anhydrase to determine picomolar to nanomolar concentrations o f free transition metal ions by fluorescence anisotropy (polarization) in a reagentless format, Carbonic anhydrase variants engineered with a cysteine replacing a residue chosen near the active site (F131C and H64C) were covalently labeled with derivatives of benzoxadiazole sulfo namide, These labeled variants exhibited changes in anisotropy up to 0 .07 upon binding free Cu(II), Co(II), and Zn(II) with apparent K-d's c lose to the values observed with wild-type apocarbonic anhydrase, The covalent attachment of the label has significant advantages over nonco valent labels we have described previously. Furthermore, the metal ion -dependent anisotropy changes were predictable using simple theory. Th e results demonstrate that free transition metal ions can be determine d at trace levels in aqueous solution using inexpensive instruments.