Study of the interaction of arsenical compounds with vicinal dithiols

CARBAJAL M. LAURA; CAUERHFF ANA; GRASSELLI MARIANO

Rosario, Santa Fe, Argentina.

Congreso; XXXV Reunión Anual de la Asociación Argentina de Biofísica.; 2006

Asociación Argentina de Biofísica

Advanced functional materials are in high demand for a variety of applications ranging from biocatalysis to chromatography, diagnostics and sensors. For this goal, it was chosen a system to immobilize recombinant proteins through binding to chemical ligands attached to polymeric materials in oriented way to improved enzymatic activity and stability.Research on the synthesis and characterization of materials were carried out using polymer modification, organo-arsenic ligands and Cys-X-X-Cys motif proteins like Thioredoxin [1]. The key point of this interaction is that it presents specificity and structural requirements. We propose to evaluate and compare the binding specificity as well as its kinetics, according to the ligand nature and the protein motif environment.The amine phenyl arsenic ligand could be show in a oxidative state (III) and (V) as oxide and acid, APAO and APA(V), respectively. The state (III) could also exists as a chlorine derivate (APACl). It is an intermediary of the synthesis reaction of the APAO so it was need to be synthesized and characterized in our lab.Our results show that the high solubility presented by the APACl as well as its chemical stability (in the range of pH among 4 and 7) allow an enhanced immobilization compared to APAO, in systems involving NHS/EDC activation.In order to a better understanding of this system, the arsenic derivates chemical reactivity to compounds with mono- and dithiols were performed by Ellman’s assay. As a result, the chemical reactivity for both compounds was similar. It was also demonstrated that the Ellman’s reagent (DTNB or 5,5´-dithio-bis(2-nitrobenzoic acid)) is capable of shifting the chemical equilibrium to the arsenic free form of both ligands. This is an important tip to have in mind for the regeneration of the arsenic activity.Kinetic and equilibrium binding analysis were performed by Biosensor technology with IAsys® Surface Plasma Resonance system (SPR). Arsenical ligand was immobilized onto carboxymethyl dextran surface via EDC/NHS chemistry. This procedure is commonly used in protein-protein interaction but it is novel with arsenical compounds. Preliminaries studies using APACl as immobilized ligand and thioredoxin as ligate, showed an association constant (kass) of 9.5x102 M-1 s-1, an equilibrium dissociation constant (KD) of 7.34x10-5 M and a ligand capacity (Rmax) of 78.63 arc seg at pH=7. However, APAO exhibited a low efficiency of immobilization due to its low solubility, presenting high trend to oxidize and to lose activity. As a conclusion, using CMD and NHS/EDC chemistry we can immobilized organo-arsenic compounds to study binding properties with proteins as soluble ligates.References: [1] “Oriented immobilization of protein on grafted porous polymers” Carbajal ML. et al., M.NIMBS, 2003, 208, 416 - 423. //Acknowledgments: M.L.C. thanks to CONICET for supporting her fellowship.