Characterization of indo-1 and quin-2 as spectroscopic probes for Zn 2+-protein interactions

Abstract

1-[2-Amino-5-(6-carboxyindol-2-yl)phenoxyl]-2-(2′ - amino - 5′ - methylphenoxy)ethane - NitN,N,N′,N′ - tetraacetic acid (indo-1) and 2-[2-(bis(carboxymethyl) amino - 5 - methylphenoxy)methyl] - 6 - methyl - 8 - [bis - (carboxymethyl)amino]quinoline (quin - 2) are sensitive, spectral indicators for Zn 2+. Additions of subsaturating Zn 2+ to 10–80 μ m indo-1 or quin-2 at pH 7.0 produce uv difference spectra with isosbestic wavelengths at 342 and 282 nm or at 342, 317, and 252 nm, respectively. Formation of 1:1 Zn 2+:indicator complexes at pH 7.0 and 20°C in the absence (presence) of 100 m m KCl gives Δϵ max = −2.4 ± 0.2 × 10 4 m −1 cm −1 at 367 nm (−2.1 ± 0.2 × 10 4 m −1 cm −1 at 365 nm) for indo-1 and Δϵ max = −2.7 ± 0.1 × 10 4 m −1 cm −1 at 266 nm (−2.6 ±0.1 × 10 4 m −1 cm −1 at 265 nm) for quin-2. Competition experiments at pH 7.0 and 20°C with indo-1 and quin-2 and also 4-(2-pyridylazo) resorcinol (PAR) as the second chelator in the absence (presence) of 100 m m KCl yield apparent affinity constants: K′ A = 2.5 ± 1.0 × 10 10 m −1 (6.2 ± 0.5 × 10 9 m −1) for indo-1 binding Zn 2+ and K′ A = 9.4 ± 3.3 × 10 11 m −1 (2.7 ± 0.1 × 10 11 m −1 for quin-2 binding Zn 2+. The above constants provide the basis for rapid steady-state spectrophotometric determinations of the affinity of a protein for Zn 2+ with K′ A ∼ 10 10–10 13 m −1. Addition of isolated regulatory dimers from Escherichia coli aspartate transcarbamoylase to excess indo-1 at pH 7.0 and 20°C, for example, gave a rapid absorbance change (<10 min) at ∼ 367 nm which was used (after correction for ∼ 20% loosely associated Zn 2+) to calculate K′ A = 1 × 10 12 m −1 (±100 m m KCl) for Zn 2+ binding to this protein. At the wavelength of maximum absorbance change with indo-1, there was little interference (<1%) by the presence of protein, free mercurial reagent, 2-mercaptoethanol, or 1 m m MgCl 2. Thus, Zn 2+ cinding constants for unstable proteins with high affinities for Zn 2+ can be measured at neutral pH by rapid equilibration with excess indo-1. With excess quin-2, the procedure must be modified to take into account the interference from protein absorbance.