title: Analysis of beryllium to biomolecule binding using a metal specific fluorescent probe and competitive assay.
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 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.
authors.affiliation: 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.
authors.forename: Yan
authors.initials: Y
authors.lastname: Zheng
authors.forename: Lin
authors.initials: L
authors.lastname: Lin
authors.forename: Wei
authors.initials: W
authors.lastname: Hang
authors.forename: Xiaomei
authors.initials: X
authors.lastname: Yan
authors.forename: Babetta L
authors.initials: BL
authors.lastname: Marrone
mesh_terms: D000255:Adenosine Triphosphate; D000818:Animals; D001608:Beryllium / Q000737:chemistry*; D001667:Binding, Competitive; D005293:Ferritins; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D009711:Nucleotides; D011485:Protein Binding; D012710:Serum Albumin, Bovine; D015032:Zinc / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D009711:Nucleotides; D012710:Serum Albumin, Bovine; D000255:Adenosine Triphosphate; D005293:Ferritins; D015032:Zinc; D001608:Beryllium