Dizinc enzyme model/complexometric indicator pairs in indicator displacement assays for inorganic phosphates under physiological conditions

A dizinc phosphohydrolase enzyme model complex employing the dinucleating ligand 2,6-bis-[(bis-pyridin-2-ylmethyl-amino)methyl]-4-methylphenol (L1) was tested for binding to a series of 11 commercially available complexometric indicators in aqueous N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer at pH 7.4, with the aim of determining the applicability of these indicators in indicator displacement assays (IDAs) under physiological conditions. Dissociation constants (Kd) were determined for 11 indicator-Zn2L1 complexes, spanning 2 orders of magnitude from 2.8 x 10(-4) M (alizarin red S) to 2.7 x 10(-6) M (bromo pyrogallol red). Phosphate and pyrophosphate were tested for their ability to displace bound indicator and produce a detectable colorimetric response. Three indicators (bromo pyrogallol red, mordant blue 9, and zincon) complex to Zn2L1 to form an indicator displacement assay selective for pyrophosphate over phosphate. Because selection of an indicator/analyte pair having appropriate relative Kd values is critical for their successful application in IDAs, the binding data for these 11 indicators should assist their extension to IDAs for other analytes.