An electrochemical-spectrophotometrical study of the oxidized forms of the mediator 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) produced by immobilized laccase

ABTS (2,2′-azino-bis-(3-ethylbenzothiazoline-6 sulfonic acid)), was characterized in an acetate buffer solution (pH 5.0) by cyclic voltammetry and thin layer spectroelectrochemistry. ABTS⁺ presented an electronic absorption spectrum with five maxima, at 214, 394, 414, 646 and 728 nm. There was no spectroscopic interference at 728 nm with ABTS or ABTS²⁺ and was the best wavelength to measure the ABTS⁺ absorbance. Also a complete oxidation of ABTS → ABTS²⁺ produced almost a colorless solution. This spectroelectrochemical observation is novel and could be extremely useful to many investigators that study ABTS-mediated oxidations catalyzed by laccase and peroxidase. The obtained results showed that enzyme laccase (fungal source: Myceliophthora thermophila) produced a reduced ABTS and stable monocation ABTS⁺ mixture with no evidence of the dication ABTS²⁺ formation. This was in agreement with the redox potential associated to this laccase. Finally the results revealed that ABTS⁺ was reduced in presence of a wide variety of organic compounds, including the interesting case of indigo blue which was completely bleached at a stoichiometric coefficient [ABTS⁺]/[Indigo] ∼ 4. These results indicate the importance of producing oxidized mediators such as ABTS ⁺, either by enzymatic or electrochemical processes in order to oxidize a wide variety of refractory compounds such as aromatic dyes and polyphenolic compounds. The fact that oxidized mediators react with the final substrate alone opens new possibilities for a wide variety of electrochemical or enzymatic reactors. The mediator recycling between enzyme and substrate is a possibility that has been shown to be feasible and those results will be published elsewhere.