Considerations about the Continuous Assay Methods, Spectrophotometric and Spectrofluorometric, of the Monophenolase Activity of Tyrosinase

• Main Authors: Pablo García-Molina, José Luis Munoz-Munoz, Joaquin A. Ortuño, José Neptuno Rodríguez-López, Pedro Antonio García-Ruiz, Francisco García-Cánovas, Francisco García-Molina
• Format: Article
• Language: English
• Published: MDPI AG 2021-08-01
• Series: Biomolecules
• Subjects: tyrosinase; polyphenol oxidase; monophenolase activity; fluorimetric method; spectrophotometric method; LOD^M
• Online Access: https://www.mdpi.com/2218-273X/11/9/1269

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 (<inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mrow><msubsup><mi mathvariant="normal">V</mi><mrow><mi>ss</mi></mrow><mrow><mi mathvariant="normal">M</mi><mo>,</mo><mi mathvariant="normal">M</mi></mrow></msubsup></mrow></semantics></math></inline-formula>) is equal to the rate of accumulation of dopachrome (<inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mrow><msubsup><mi mathvariant="normal">V</mi><mrow><mi>ss</mi></mrow><mrow><mi mathvariant="normal">M</mi><mo>,</mo><mi>DC</mi></mrow></msubsup></mrow></semantics></math></inline-formula>) or to the rate of accumulation of its oxidized adduct, originated by the nucleophilic attack on <i>o</i>-quinone by a nucleophile such as 3-methyl-2-benzothiazolinone (MBTH), (<inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mrow><msubsup><mi mathvariant="normal">V</mi><mrow><mi>ss</mi></mrow><mrow><mi mathvariant="normal">M</mi><mo>,</mo><mrow><mtext> </mtext><mi mathvariant="normal">A</mi></mrow><mo>−</mo><mi>ox</mi></mrow></msubsup></mrow></semantics></math></inline-formula>), 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 LOD^M (limit of detection of the monophenolase activity) values with spectrophotometric methods. The spectrofluorimetric methods had a lower LOD^M than spectrophotometric methods. In the case of 4-hydroxyphenil-propionic acid, the LOD^M obtained by us was 0.25 U/mL. Considering the relative sensitivities of 4-hydroxyanisole, compared with 4-hydroxyphenil-propionic acid, LOD^M values like those obtained by fluorescent methods would be expected.