RESUMO
A new pulse UV irradiation-induced chemiluminescence (CL) determination method was developed for l-tyrosine using the luminol derivative L-012. The proposed method depends on the formation of reactive oxygen species (ROS) upon pulse UV irradiation of l-tyrosine; then, these ROS react with L-012 producing strong CL. The proposed method showed excellent sensitivity and ultraselectivity toward l-tyrosine. The mechanism of the developed CL method was studied using ROS scavengers, HPLC, and mass spectrometry. The method was linear for l-tyrosine in the range of 0.03-50 µM. Minor changes in the l-tyrosine structure, including hydroxylation, dehydroxylation, phosphorylation, or decarboxylation, were found to lead to a strong decrease in CL. Using the excellent selectivity of the proposed method for l-tyrosine, we have developed a CL assay for measuring alkaline phosphatase activity in the range of 0.02-15 U/L with the limit of detection (LOD) of 4 mU/L using the nonchemiluminescent O-phospho-l-tyrosine as a substrate. Furthermore, the CL reaction was applied for tyrosinase activity assay as this enzyme can convert l-tyrosine to the nonchemiluminescent l-dopa. The decrease in CL is correlated with the tyrosinase activity in the range of 0.025-0.75 U/mL with an LOD of 1.5 mU/mL. Moreover, the tyrosinase activity assay was successfully applied for the determination of IC50 of the tyrosinase inhibitors kojic acid and benzoic acid. Therefore, our novel pulse UV irradiation CL method for the determination of l-tyrosine was not only suitable for the determination of this vital amino acid but also extended to the successful determination of its producing and metabolizing enzymes and their inhibitors.