Kayllane Vitória Brito Lopes¹, Francisco Eroni P. dos Santos¹, Cícero Alves Lopes Júnior¹

The transport of substances within the human body is facilitated by reversible binding to human serum albumin (HSA). This reversible interaction has been demonstrated to enhance the solubility of hydrophobic drugs, thereby influencing their circulation, metabolism, and therapeutic efficacy. Due to its substantial homology (>70%) with HSA and analogous binding behavior, bovine serum albumin (BSA) is frequently utilized in in vitro studies. In some pharmaceutical formulations, azo dyes are used as excipients, but studies show that BSA undergoes conformational changes upon interaction with these dyes and low molecular weight drugs, affecting its secondary and tertiary structures. These interactions have the capacity to influence the absorption, distribution, metabolism, and toxicity of dyes, as well as to modify the structure and function of HSA. Consequently, the investigation of drug-protein interactions is imperative for the domains of pharmacokinetics and toxicology, as it serves to substantiate the effectiveness of pharmaceutical agents. Consequently, this study evaluated the complex formed by protein, drug, and dye by UV-Vis and molecular fluorescence. A comprehensive evaluation was conducted, encompassing several parameters, including pH (5.5–8.0) and temperature (25–35 °C), along the type of azo dyes (tartrazine and amaranth). The dyes exhibited consistent absorptivity within the evaluated pH and temperature ranges. The effects of pH and temperature were more evident for cephalexin at low concentrations, especially at pH 5.5 and 8.0, due to its susceptibility to acid and alkaline hydrolysis. The BSA demonstrated heightened sensitivity to the conditions of the medium, particularly at pH 5.5, which is in proximity to its isoelectric point (~4.7), and at pH 8.0, a level that promoted aggregation and precipitation. In order to study the interactions in the protein-drug-dye system, the concentration of one of the components was varied while the others remained constant. The working ranges that were established included dyes at concentrations ranging from 10 to 80 µmol/L, BSA at concentrations ranging from 5 to 20 µmol/L, and cephalexin at concentrations ranging from 150 to 600 µmol/L. The fixed concentrations of the reagents employed in this study were as follows: 50 µmol/L of dye, 20 µmol/L of protein, and 250 µmol/L of drug. The UV-Vis spectra indicated the formation of the complex through an increase in absorbance. When the concentration of cephalexin was varied, an interaction with BSA was observed. Preliminary fluorescence data demonstrated that tartrazine functions as a fluorescence quencher. A slight blue shift in emission wavelengths indicated that the BSA fluorophore was in a more hydrophobic environment after the addition of tartrazine. Subsequent studies will endeavor to optimize and further develop the application of fluorescence spectroscopy to enhance comprehension of the impact of the two dyes on binding and interaction within the study complex. The calculation of binding constants and thermodynamic parameters from the data will contribute to a more comprehensive understanding of the system studied. These results may provide novel insights into the impact of azo dyes on the binding mechanism of drugs to albumin, offering novel perspectives on how these dyes can influence the therapeutic efficacy of drugs and raising concerns about the potent health risks associated with the use of azo dyes in pharmaceutical formulations.

Agradecimentos: UFPI, CNPq.