Considerations about the Continuous Assay Methods, Spectrophotometric and Spectrofluorometric, of the Monophenolase Activity of Tyrosinase
Pablo García-Molina,
José Luis Munoz-Munoz,
Joaquin A. Ortuño,
José Neptuno Rodríguez-López,
Pedro Antonio García-Ruiz,
Francisco García-Cánovas,
Francisco García-Molina
Affiliations
Pablo García-Molina
GENZ-Group of Research on Enzymology, Department of Biochemistry and Molecular Biology-A, Regional Campus of International Excellence “Campus Mare Nostrum”, University of Murcia, Espinardo, 30100 Murcia, Spain
José Luis Munoz-Munoz
Microbial Enzymology Group, Department of Applied Sciences, University of Northumbria, Ellison Building A, Newcastle Upon Tyne NE1 8ST, UK
Joaquin A. Ortuño
Department of Analytical Chemistry, Faculty of Chemistry, University of Murcia, 30100 Murcia, Spain
José Neptuno Rodríguez-López
GENZ-Group of Research on Enzymology, Department of Biochemistry and Molecular Biology-A, Regional Campus of International Excellence “Campus Mare Nostrum”, University of Murcia, Espinardo, 30100 Murcia, Spain
Pedro Antonio García-Ruiz
Group of Chemistry of Carbohydrates, Industrial Polymers and Additives, Department of Organic Chemistry, Regional Campus of International Excellence “Campus Mare Nostrum”, University of Murcia, Espinardo, 30100 Murcia, Spain
Francisco García-Cánovas
GENZ-Group of Research on Enzymology, Department of Biochemistry and Molecular Biology-A, Regional Campus of International Excellence “Campus Mare Nostrum”, University of Murcia, Espinardo, 30100 Murcia, Spain
Francisco García-Molina
Department of Anatomía Patológica, Hospital General Universitario Reina Sofía, Av. Intendente Jorge Palacios, 1, 30003 Murcia, Spain
With the purpose to obtain the more useful tyrosinase assay for the monophenolase activity of tyrosinase between the spectrofluorometric and spectrophotometric continuous assays, simulated assays were made by means of numerical integration of the equations that characterize the mechanism of monophenolase activity. These assays showed that the rate of disappearance of monophenol (VssM,M) is equal to the rate of accumulation of dopachrome (VssM,DC) or to the rate of accumulation of its oxidized adduct, originated by the nucleophilic attack on o-quinone by a nucleophile such as 3-methyl-2-benzothiazolinone (MBTH), (VssM, A−ox), despite the existence of coupled reactions. It is shown that the spectrophotometric methods that use MBTH are more useful, as they do not have the restrictions of the L-tyrosine disappearance measurement method, of working at pH = 8 and not having a linear response from 100 μM of L-tyrosine. It is possible to obtain low LODM (limit of detection of the monophenolase activity) values with spectrophotometric methods. The spectrofluorimetric methods had a lower LODM than spectrophotometric methods. In the case of 4-hydroxyphenil-propionic acid, the LODM obtained by us was 0.25 U/mL. Considering the relative sensitivities of 4-hydroxyanisole, compared with 4-hydroxyphenil-propionic acid, LODM values like those obtained by fluorescent methods would be expected.
