Estudo eletroquímico e eletroanalítico dos biomarcadores de doenças humanas 7-metil-guanosina, orto tirosina e 3-nitro-tirosina

Abstract

Oxidative damage in biological molecules, such as double-stranded DNA (dsDNA) and proteins are associated with numerous pathologies in the human body, such as cancer. Thus, it is of great relevance to investigate the redox mechanisms that occur in vivo for different biological processes. The present study aims to investigate the redox behavior of the biomarkers of human diseases, 7-methyl-guanosine (7-mGuo), ortho-tyrosina (o-Tyr) and 3-nitro-tyrosine (3-NO2-Tyr) on glassy carbon electrode (GCE) using voltammetric techniques, such as cyclic voltammetry (CV), square wave voltammetry (SWV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS). The electrochemical results of o-Tyr and 3-NO2-Tyr were also compared with the para-tyrosine (p-Tyr). Electroanalytical methodologies for the detection and quantification of these species were also investigated and developed in the absence and presence of possible interferences. Oxidation of the 7-mGuo on GCE occurs in single step pH-dependent irreversible pathway with the transfer of one electron and one proton with formation of polymer products absorbing on the electrode surface. An oxidation mechanism of 7-mGuo has been proposed. An electroanalytical method for quantification of 7-mGuo at physiological pH, using GCE and DPV was proposed, with a detection limit of 3.26 μmol L-1 and limit of quantification of 10,88 μmol L-1. Electrochemical detection of 7-mGuo was also investigated in the presence of potential interference of guanine, guanosine and 7-methylguanine (7-mGua). The study showed that the anodic peak of 7-mGuo suffered no interference of these species, since oxidation occurs in a very different potential. In general, the p- and o-Tyr undergo oxidation in a single irreversible step pH-dependent, with the transfer of one electron and one proton, from the phenolic group to formation of Tyr phenoxy radical (Tyr●). However, while the p-Tyr● radical preferably polymerizes, forming a resistive film on the GCE surface, the o-Tyr● reacts preferentially with water with formation of o- and p-quinone derivatives, that are adsorbed and reversibly reduced on the GCE surface. In relation the 3-NO2-Tyr its oxidation occurs, in general, in two irreversible steps. The first step is pH-dependent, while the second is pH-independent, indicating the absence of protons in the process. The first process correspond to the oxidation of the phenolic group to form 3-NO2-Tyr●, which reacts in different ways, polymerizing, forming a resistive film on the GCE surface and/or being directly electro-oxidized to a cationic product (second step). The voltammetric data also showed that the 3-NO2-Tyr phenol group is more difficult to oxidize when compared to p- and o-Tyr molecules. Moreover, unlike p-Tyr and o-Tyr that present no cathodic peak, 3-NO2-Tyr suffers in acid medium electro-reduction in a single irreversible step with formation of two electroactive products. Such processes were assigned to the reduction of the nitro group to form hydroxylamine and amine. Thus, is clearly demonstrated that the nitro group attached, as well as the phenolic group position at the Tyr molecule, strongly influence its redox properties. The redox mechanism of o-Tyr and 3-NO2-Tyr are presented and discussed. A voltammetric method for detection and quantification of 3-NO2-Tyr in physiological medium using DPV was also proposed and a concentration range of 20 to 200 μmol L-1, presented a correlation coefficient of 0.998 and a detection limit of 6, 21 μmol L-1. The knowledge of redox mechanisms of the biomarkers of human diseases, 7-mGuo, o-Tyr and 3-NO2-Tyr, as well as the proposed electroanalytical methods for their quantification correlates and are important in the literature, as basic knowledge to future interpretation and applications in molecular biochemistry.