Characterization of indo-1 and quin-2 as spectroscopic probes for Zn2(+)-protein interactions.
1-[2-Amino-5-(6-carboxyindol-2-yl)phenoxyl]-2-(2'- amino-5'-methylphenoxy)ethane-N,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 microM 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 degrees C in the absence (presence) of 100 mM KCl gives delta epsilon max = -2.4 +/- 0.2 X 10(4) M-1 cm-1 at 367 nm (-2.1 +/- 0.2 X 10(4) M-1 cm-1 at 365 nm) for indo-1 and delta epsilon max = -2.7 +/- 0.1 X 10(4) M-1 cm-1 at 266 nm (-2.6 +/- 0.1 X 10(4) M-1 cm-1 at 265 nm) for quin-2. Competition experiments at pH 7.0 and 20 degrees 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 X 10(10) M-1 (6.2 +/- 0.5 X 10(9) M-1) for indo-1 binding Zn2+ and K'A = 9.4 +/- 3.3 X 10(11) M-1 (2.7 +/- 0.1 X 10(11) M-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 approximately 10(10) - 10(13) M-1.(ABSTRACT TRUNCATED AT 250 WORDS)
Jefferson
J R
JR
Section on Protein Chemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Hunt
J B
JB
Ginsburg
A
A
D000634:Aminoquinolines; D001221:Aspartate Carbamoyltransferase / Q000032:analysis; D001667:Binding, Competitive; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins / Q000032:analysis*; D004926:Escherichia coli / Q000201:enzymology; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D007211:Indoles; D008667:Metalloproteins / Q000032:analysis*; D012680:Sensitivity and Specificity; D013053:Spectrophotometry; D015032:Zinc / Q000032:analysis*
D000634:Aminoquinolines; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D007211:Indoles; D008667:Metalloproteins; D001221:Aspartate Carbamoyltransferase; D015032:Zinc; C048960:indo-1; C035739:Quin2