Molar absorption coefficients and stability constants of metal complexes of 4-(2-pyridylazo)resorcinol (PAR): Revisiting common chelating probe for the study of metalloproteins

Abstract

4-(2-Pyridylazo)resorcinol (PAR) is one of the most popular chromogenic chelator used in the determination of the concentrations of various metal ions from the d, p and f blocks and their affinities for metal ion-binding biomolecules. The most important characteristics of such a sensor are the molar absorption coefficient and the metal–ligand complex dissociation constant. However, it must be remembered that these values are dependent on the specific experimental conditions (e.g. pH, solvent components, and reactant ratios). If one uses these values to process data obtained in different conditions, the final result can be under- or overestimated. We aimed to establish the spectral properties and the stability of PAR and its complexes accurately with Zn2+, Cd2+, Hg2+, Co2+, Ni2+, Cu2+, Mn2+ and Pb2+ at a multiple pH values. The obtained results account for the presence of different species of metal–PAR complexes in the physiological pH range of 5 to 8 and have been frequently neglected in previous studies. The effective molar absorption coefficient at 492nm for the ZnHx(PAR)2 complex at pH7.4 in buffered water solution is 71,500M−1cm−1, and the dissociation constant of the complex in these conditions is 7.08×10−13M2. To confirm these values and estimate the range of the dissociation constants of zinc-binding biomolecules that can be measured using PAR, we performed several titrations of zinc finger peptides and zinc chelators. Taken together, our results provide the updated parameters that are applicable to any experiment conducted using inexpensive and commercially available PAR.