Colorimetric and Fluorimetric Assays to Quantitate Micromolar Concentrations of Transition Metals

Transition metal ions, although maintained at low concentrations, play diverse important roles in many biological processes. Two assays useful for the rapid quantification of a range of first-row transition metal ions have been developed. The colorimetric assay extends the 4-(2-pyridylazo)resorcinol assay of Hunt et al. (J. Biol. Chem. 255, 14793 (1984)) to measure nanomole quantities of Co2+, Ni2+, and Cu2+ as well as Zn2+. The fluorimetric assay takes advantage of the coordination of a number of metal ions (Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+) by Fura-2 and can also be used to measure nanomole quantities of these ions. The assays developed here have the advantage of not requiring the extensive sample preparation necessary for other methodologies, such as atomic absorption spectroscopy and inductively coupled plasma emission spectroscopy (ICPES), while being comparable in accuracy to the detection limits of ICPES for the first-row transition metal ions. To demonstrate the effectiveness of these assays, we determined the affinity of carbonic anhydrase II (CA II), a prototypical zinc enzyme, for Ni2+ and Cd2+. These data indicate that CA II binds transition metals with high affinity and is much more selective for Zn2+ over Ni2+ or Cd2+ than most small-molecule chelators or other metalloenzymes.