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.
Arsenic carcinogenesis Circular dichroism Conformational studies Drug delivery systems Thioredoxin
Compound between p-aminophenylarsenoxide and fluorescein isothiocyanate
Attenuated total reflectance-fourier transform infrared
Phosphate saline buffer
Relative fluorescence units
Sodium dodecyl sulfate polyacrylamide gel electrophoresis
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We want to thank Dr. Gerardo Caballero for helpful discussions with mass spectra analyses, and to Lic Martín Noguera for his assistance with CD experiments. This work was supported by grants from Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET; PIP 5832/05) and Universidad Nacional de Quilmes (UNQ; UNQ-MPBio 53/1001(PUNQ0396/07), MINCyT (PME 01730). S del VA, MLC and MG are members of CONICET (Scientific Research Program) and IMBICE. Fellowships for ALF (CONICET, Posgrado T-I and II, D. No. Res. 1086/09), CFT (CONICET, Posgrado T-I and II, D. No. Res. 3100/08) and MSA (UNQ, No. Res. (CS) 113/07) are acknowledged.