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

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Abstract

The metallochromic indicator 4-(2-pyridylazo)resorcinol (PAR) has been used at pH 7.0 to monitor the mercurial-promoted Zn2+ release from Escherichia coli aspartate transcarbamoylase and Zn2+ uptake by regulatory dimers upon displacement of the mercurial reagent with 2-mercaptoethanol. The release of Zn2+ (as reflected by a yellow to orange color change in PAR solutions) is linked to dissociation of the enzyme since the six Zn2+ bonding domains stabilize catalytic and regulatory chain contacts; the rebinding of Zn2+ produces enzyme assembly and a corresponding decrease in the amount of PAR-Zn2+ complex. Using 10-fold PAR to free Zn2+ at pH 7.0, Δϵ = 6.6 ± 0.2 × 104m−1 cm−1 at 500 nm (20°C) for (PAR)2Zn2+ complex formation (β2 ≅ 1012m−1). In kinetic studies at pH 7.0, PAR (10−4m) has been used to measure the instantaneous concentration of Zn2+ released from micromolar quantities of protein; second-order k = 2 × 107m−1 s−1 for forming the 1:1 PAR:Zn2+ complex. These properties of PAR-Zn2+ interactions make PAR a generally useful reagent for studying Zn2+ release from proteins.

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    This work was performed under Title IV, Intergovernmental Personnel Act.

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    Present address: Chemistry Department, The Catholic University of America, Washington, D. C. 20064.

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