A continuous spectrophotometric assay for determination of the aureusidin synthase activity of tyrosinase

Abstract

Introduction – Aurones (aureusidin glycosides) are plant flavonoids that provide yellow colour to the flowers of some orna-mental plants. In this study we analyse the capacity of tyrosinase to catalyse the synthesis of aureusidin by tyrosinase fromthe chalcone THC (2′,4′,6′,4–tetrahydroxychalcone).Objective – To develop a simple continuous spectrophotometric assay for the analysis of the spectrophotometric and kineticcharacteristics of THC oxidation by tyrosinase.Methodology – THC oxidation was routinely assayed by measuring the increase in absorbance at 415 nm vs. reaction time.Results – According to the mechanism proposed for tyrosinase, the enzymatic reaction involves the o-hydroxylation of themonophenol THC to the o-diphenol (PHC, 2′,4′,6′,3,4 – pentahydroxychalcone), which is then oxidised to the correspondingo-quinone in a second enzymatic step. This product is highly unstable and thus undergoes a series of fast chemical reactionsto produce aureusidin. In these experimental conditions, the optimum pH for THC oxidation is 4.5. The progress curvesobtained for THC oxidation showed the appearance of a lag period. The following kinetic parameters were also determined:Km = 0.12 mM , Vm = 13 mM /min, Vm/Km = 0.11/min.Conclusion – This method has made it possible to analyse the spectrophotometric and kinetic characteristics of THC by tyro-sinase. This procedure has the advantages of a short analysis time, straightforward measurement techniques and repro-ducibility. In addition, it also allows the study of tyrosinase inhibitors, such as tropolone.