Department of Chemical Biology, College of Chemistry and Chemical Engineering, The Key Laboratory for Chemical Biology of Fujian Province, The Key Laboratory of Analytical Science, Xiamen University, Xiamen 361005, China.
Talanta. 2011 Jul 15;85(1):638-43. doi: 10.1016/j.talanta.2011.04.037. Epub 2011 Apr 16.
Studying metal-biomolecule interactions is critical to the elucidation of the molecular basis of the biological functions and toxicity of metals. In the present study, a competitive fluorimetric approach has been developed to measure the apparent affinity of biomolecules for Be(2+) by using a Be(2+)-specific fluorigenic probe (10-hydroxybenzo[h]quinoline-7-sulfonate, HBQS). Under physiological conditions, HBQS coordinates with Be(2+) in a molar ratio of 1:1 and results in a fluorescence shift from 580 nm for HBQS to 480 nm for the Be-HBQS complex associated with significant fluorescence enhancement. When a beryllium ligand is present in the mixture of Be(2+) and HBQS, the competition of ligand against HBQS for beryllium ion binding results in dissociation and thus a fluorescence decrease of the Be-HBQS complex. By titrating ligand and monitoring the dose-dependent decrease of Be-HBQS complex fluorescence at 480 nm, the apparent affinity between ligand and Be(2+) can be derived. Applying this simple approach, the apparent affinities of various nucleotides and the iron-storage protein ferritin for beryllium ion have been determined. In particular, the apparent dissociation constant of Be(2+) and adenosine 5'-triphosphate (ATP) was also validated by an electrospray ionization mass spectrometric (ESI-MS) method. The general applicability of the proposed competition assay was further demonstrated using FluoZin-1, a zinc fluorescent indicator, in a binding study for Zn(2+) and bovine serum albumin. This newly developed competitive fluorimetric assay provides a sensitive, simple, and generic approach for affinity estimation of metal and biomolecule binding.
研究金属-生物分子相互作用对于阐明金属的生物功能和毒性的分子基础至关重要。在本研究中，开发了一种竞争荧光测定方法，通过使用铍（Be（2+））特异性荧光探针（10-羟基苯并[h]喹啉-7-磺酸，HBQS）来测量生物分子对Be（2+）的表观亲和力。在生理条件下，HBQS 以 1:1 的摩尔比与 Be（2+）配位，导致荧光从 HBQS 的 580nm 转移到与 Be-HBQS 复合物相关的 480nm，同时伴随着显著的荧光增强。当混合物中存在铍配体时，配体与 HBQS 竞争结合铍离子，导致 Be-HBQS 复合物解离，从而荧光降低。通过滴定配体并监测 480nm 处 Be-HBQS 复合物荧光的剂量依赖性降低，可以得出配体与 Be（2+）之间的表观亲和力。应用这种简单的方法，已经确定了各种核苷酸和铁储存蛋白 ferritin 与铍离子的表观亲和力。特别是，Be（2+）和腺苷 5'-三磷酸（ATP）的表观离解常数也通过电喷雾电离质谱（ESI-MS）方法进行了验证。在锌荧光指示剂 FluoZin-1 用于结合研究 Zn（2+）和牛血清白蛋白的实验中，进一步证明了所提出的竞争测定法的通用性。这种新开发的竞争荧光测定法为金属和生物分子结合的亲和力估计提供了一种灵敏、简单和通用的方法。
