A direct spectrophotometric method for the simultaneous determination of zinc and cobalt in metalloproteins using 4-(2-pyridylazo)resorcinol.

An assay involving the direct and simultaneous determination of low micromolar concentrations (1-10 microM) of both zinc and cobalt ions suitable for metal content analyses of metalloproteins is described. The procedure exploits differences in the visible absorption spectra of the chromophoric chelator 4-(2-pyridylazo)resorcinol (PAR) resulting from its complexation to Zn2+ and/or Co2+ ions and is based on the fit of experimental spectra to a linear addition of Beer-Lambert law. The method eliminates the need for separating or masking one of the metal ions prior to their quantification and could prove to be particularly useful in studies on Co2+-substituted zinc proteins.