The use of 4-(2-pyridylazo)resorcinol in studies of zinc release from Escherichia coli aspartate transcarbamoylase

The metallochromic indicator 4-(2-pyridylazo)resorcinol (PAR) has been used at pH 7.0 to monitor the mercurial-promoted Zn 2+ release from Escherichia coli aspartate transcarbamoylase and Zn 2+ uptake by regulatory dimers upon displacement of the mercurial reagent with 2-mercaptoethanol. The release of Zn 2+ (as reflected by a yellow to orange color change in PAR solutions) is linked to dissociation of the enzyme since the six Zn 2+ bonding domains stabilize catalytic and regulatory chain contacts; the rebinding of Zn 2+ produces enzyme assembly and a corresponding decrease in the amount of PAR-Zn 2+ complex. Using 10-fold PAR to free Zn 2+ at pH 7.0, Δϵ = 6.6 ± 0.2 × 10 4 m −1 cm −1 at 500 nm (20°C) for (PAR) 2Zn 2+ complex formation (β 2 ′ ≅ 10 12 m −1). In kinetic studies at pH 7.0, PAR (10 −4 m) has been used to measure the instantaneous concentration of Zn 2+ released from micromolar quantities of protein; second-order k = 2 × 10 7 m −1 s −1 for forming the 1:1 PAR:Zn 2+ complex. These properties of PAR-Zn 2+ interactions make PAR a generally useful reagent for studying Zn 2+ release from proteins.