An Arsenic Fluorescent Compound as a Novel Probe to Study Arsenic-Binding Proteins

Abstract

Arsenic-binding proteins are under continuous research. Their identification and the elucidation of arsenic/protein interaction mechanisms are important because the biological effects of these complexes may be related not only to arsenic but also to the arsenic/protein structure. Although many proteins bearing a CXXC motif have been found to bind arsenic in vivo, new tools are necessary to identify new arsenic targets and allow research on protein/arsenic complexes. In this work, we analyzed the performance of the fluorescent compound APAO-FITC (synthesized from p-aminophenylarsenoxide, APAO, and fluorescein isothiocyanate, FITC) in arsenic/protein binding assays using thioredoxin 1 (Trx) as an arsenic-binding protein model. The Trx-APAO-FITC complex was studied through different spectroscopic techniques involving UV-Vis, fluorescence, atomic absorption, infrared and circular dichroism. Our results show that APAO-FITC binds efficiently and specifically to the Trx binding site, labeling the protein fluorescently, without altering its structure and activity. In summary, we were able to study a protein/arsenic complex model, using APAO-FITC as a labeling probe. The use of APAO-FITC in the identification of different protein and cell targets, as well as in in vivo biodistribution studies, conformational studies of arsenic-binding proteins, and studies for the design of drug delivery systems for arsenic anti-cancer therapies, is highly promising.

Therapeutic and analytical applications of arsenic binding to proteins

“…It has been demonstrated that binding to PAO does not induce alterations in the structure of Trx. 103…”

Recognition of Proteins by Metal Chelation-Based Fluorescent Probes in Cells

“…Besides these EDT-containing molecules, a similar As(III)-based probe, APAO-FITC, was synthesized with only one As-based moiety with the EDT being replaced by two hydroxyl groups (-OH). APAO-FITC has been shown to label native As-binding proteins without strong disturbance on their secondary structures (Femia et al, 2012). To expand the toolbox of As(III)-based probes, the biotin-avidin system was then taken into consideration while the As(III)-binding proteins could be analyzed by the combination of proteome microarray assay with biotin-conjugated As, i.e., Biotin-As.…”

“…The first generation of As(III)-based fluorescent probes, FLAsH-EDT 2 and ReAsH-EDT 2 , mainly target proteins with genetically fused tetracysteine tag since this motif seems to be rare among native proteins (Femia et al, 2012). Moreover, two As-based moieties of the probes result in stable protein labeling, and these two probes work successfully in the visualization of intracellular proteins as well as in affinity chromatography to track arsenic-binding proteins (Griffin et al, 1998).…”

Elucidating arsenic-bound proteins in the protein data bank: data mining and amino acid cross-validation through Raman spectroscopy

“…7,22 By continuously elucidating the structures of arsenic-binding proteins, it is possible to understand how arsenic affects cells. 31 Various data from biomolecular studies show that arsenic binding to macromolecules causes heavy metal sequestration in bacterial proteins. However, the precise mechanism of arsenic transfer into human cells is currently unknown.…”