Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10559849 | Talanta | 2011 | 6 Pages |
Abstract
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 Be2+ by using a Be2+-specific fluorigenic probe (10-hydroxybenzo[h]quinoline-7-sulfonate, HBQS). Under physiological conditions, HBQS coordinates with Be2+ in a molar ratio of 1:1 and results in a fluorescence shift from 580Â nm for HBQS to 480Â nm for the Be-HBQS complex associated with significant fluorescence enhancement. When a beryllium ligand is present in the mixture of Be2+ 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 480Â nm, the apparent affinity between ligand and Be2+ 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 Be2+ and adenosine 5â²-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 Zn2+ 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.
Related Topics
Physical Sciences and Engineering
Chemistry
Analytical Chemistry
Authors
Yan Zheng, Lin Lin, Wei Hang, Xiaomei Yan, Babetta L. Marrone,