CHARACTERIZATION OF INDO-1 AND QUIN-2 AS SPECTROSCOPIC PROBES FOR ZN-2+-PROTEIN INTERACTIONS
CHARACTERIZATION OF INDO-1 AND QUIN-2 AS SPECTROSCOPIC PROBES FOR ZN-2+-PROTEIN INTERACTIONS
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DOI：
10.1016/0003-2697(90)90465-l
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发表时间：
1990-06-01
影响因子：
2.9
通讯作者：
GINSBURG, A
中科院分区：
文献类型：
--
作者：
JEFFERSON, JR;HUNT, JB;GINSBURG, A
1-[2-氨基-5-（6-羧基吲哚-2-基）苯氧基]-2-（2“-氨基-5”-甲基苯氧基）乙烷-N，N，N“，N”-四乙酸（indo-1）和2-[2-（双（羧甲基）氨基-5-甲基苯氧基）甲基]-6-甲基-8-[双（羧甲基）氨基]喹啉（quin-2）是Zn ~（2+）的灵敏光谱指示剂。在pH 7.0下，将亚饱和Zn 2+加入到10-80 μ M indo-1或quin-2中，产生分别在342和282 nm或342、317和252 nm处具有等吸光波长的UV差光谱。在pH7.0和20 ℃下，在不存在（存在）100 mM KCl的情况下形成1：1的Zn 2+：指示剂复合物，得到Δ。最大值= -2.4 .+-。0.2. times. 104 M-1 cm-1（367 nm）（-2.1 . ±. 0.2. times. 104 M-1 cm-1，在365 nm处）。最大值= -2.7 .+-。0.1. times. 104 m-1 cm-1（-2.6 . ±. 0.1. times. 104 M-1 cm-1，265 nm）。在pH7.0和20 ℃下，用indo-1和quin-2以及4-（2-吡啶偶氮）间苯二酚（PAR）作为第二螯合剂，在不存在（存在）100 mM KCl的条件下进行竞争实验，得到表观亲和常数：KA“= 2.5 × 1.0。1010 M-1（6.2 . ±. 0.5. times. 109 M-1），KA“= 9.4 ±-。3.3. times. 1011 M-1（2.7 .+-. 0.1. times. 1011M-1）。上述常数提供了快速稳态分光光度法测定蛋白质对Zn 2+与KA的亲和力的基础。1010 - 1013 M-1。例如，在pH7.0和20 ℃下，向过量的indo-1中加入从大肠杆菌谷氨酸转氨甲酰酶分离的调节二聚体，在λ下产生快速的吸光度变化（< 10分钟）。367 nm，其被使用（在校正apx. 20%松散缔合的Zn 2+），以计算KA“= 1 ×1012 M-1（. ±. 100 mM KCl）用于Zn 2+与该蛋白结合。在indo-1的最大吸光度变化波长处，蛋白质、游离汞试剂、2-巯基乙醇或1 mM MgCl 2的存在几乎没有干扰（< 1%）。因此，Zn 2+结合常数与Zn 2+的高亲和力不稳定的蛋白质，可以在中性pH值下通过快速平衡过量的indo-1。在过量quin-2的情况下，必须修改程序以考虑来自蛋白质吸光度的干扰。
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 .mu.M indo-1 or quin-2 at pH 7.0 produce uv difference spectra with isosbestic wave lengths 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.degree.C in the absence (presence) of 100 mM KC1 gives .DELTA..epsilon.max = -2.4 .+-. 0.2 .times. 104 M-1 cm-1 at 367 nm (-2.1 .+-. 0.2 .times. 104 M-1 cm-1 at 365 nm) for indo-1 and .DELTA..epsilon.max = -2.7 .+-. 0.1 .times. 104 m-1 cm-1 at 266 nm (-2.6 .+-. 0.1 .times. 104 M-1 cm-1 at 265 nm) for quin-2. Competition experiments at pH 7.0 and 20.degree.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: KA'' = 2.5 1.0 .times. 1010 M-1 (6.2 .+-. 0.5 .times. 109 M-1) for indo-1 binding Zn2+ and KA'' = 9.4 .+-. 3.3 .times. 1011 M-1 (2.7 .+-. 0.1 .times. 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 KA'' .apprx. 1010 - 1013 M-1. Addition of isolated regulatory dimers from Escherichia coli asparatate transcarbamoylase to excess indo-1 at pH 7.0 and 20.degree.C, for example, gave a rapid absorbance change (< 10 min) at .apprx. 367 nm which was used (after correction for .apprx. 20% loosely associated Zn2+) to calculate KA'' = 1 .times. 1012 M-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+ binding 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.