Characterization of indo-1 and quin-2 as spectroscopic probes for Zn2+-protein interactions

https://doi.org/10.1016/0003-2697(90)90465-LGet rights and content

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 Zn2+. Additions of subsaturating Zn2+ to 10–80 μm 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 Zn2+:indicator complexes at pH 7.0 and 20°C in the absence (presence) of 100 mm KCl gives Δϵmax = −2.4 ± 0.2 × 104m−1 cm−1 at 367 nm (−2.1 ± 0.2 × 104m−1 cm−1 at 365 nm) for indo-1 and Δϵmax = −2.7 ± 0.1 × 104m−1 cm−1 at 266 nm (−2.6 ±0.1 × 104m−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 mm KCl yield apparent affinity constants: K′A = 2.5 ± 1.0 × 1010m−1 (6.2 ± 0.5 × 109m−1) for indo-1 binding Zn2+ and K′A = 9.4 ± 3.3 × 1011m−1 (2.7 ± 0.1 × 1011m−1 for quin-2 binding Zn2+. The above constants provide the basis for rapid steady-state spectrophotometric determinations of the affinity of a protein for Zn2+ with K′A ∼ 1010–1013m−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 Zn2+) to calculate K′A = 1 × 1012m−1 (±100 mm KCl) for Zn2+ 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 mm MgCl2. Thus, Zn2+ cinding constants for unstable proteins with high affinities for Zn2+ 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.

References (22)

  • J.B. Hunt et al.

    J. Biol. Chem

    (1984)
  • J.B. Hunt et al.

    Anal. Biochem

    (1985)
  • J.P. Rosenbusch et al.

    ProBiol. Chem

    (1971)
  • K.H. Kim et al.

    J. Mol. Biol

    (1987)
  • G. Grynkiewicz et al.

    J. Biol. Chem

    (1985)
  • T.R. Hesketh et al.

    J. Biol. Chem

    (1983)
  • A. Klug et al.

    Trends Biochem. Sci

    (1987)
  • Y.R. Yang et al.
  • A. Shrake et al.

    J. Biol. Chem

    (1981)
  • A.P. Jackson et al.

    FEBS Lett

    (1987)
  • J.P. Rosenbusch et al.
  • Cited by (0)

    Presented in part at the 1988 FASEB meeting in Las Vegas, Nevada [Jefferson, J. R., Hunt, J. B., and Ginsburg, A. (1988) FASEB J. 2(5), A1338].

    2

    Present address: University of Cincinnati, College of Pharmacy, Cincinnati, OH 45267.

    3

    Present address: National Science Foundation, 1800 G Street, N.W., Washington, DC 20550.

    View full text