A continuous spectrophotometric assay for determination of the aureusidin synthase activity of tyrosinase.
BACKGROUND
Aurones (aureusidin glycosides) are plant flavonoids that provide yellow colour to the flowers of some ornamental plants. In this study we analyse the capacity of tyrosinase to catalyse the synthesis of aureusidin by tyrosinase from the chalcone THC (2',4',6',4-tetrahydroxychalcone).


OBJECTIVE
To develop a simple continuous spectrophotometric assay for the analysis of the spectrophotometric and kinetic characteristics of THC oxidation by tyrosinase.


METHODS
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 the monophenol THC to the o-diphenol (PHC, 2',4',6',3,4 - pentahydroxychalcone), which is then oxidised to the corresponding o-quinone in a second enzymatic step. This product is highly unstable and thus undergoes a series of fast chemical reactions to produce aureusidin. In these experimental conditions, the optimum pH for THC oxidation is 4.5. The progress curves obtained for THC oxidation showed the appearance of a lag period. The following kinetic parameters were also determined: K(m )= 0.12 mM, V(m )= 13 microM/min, V(m)/K(m )= 0.11/min.


CONCLUSIONS
This method has made it possible to analyse the spectrophotometric and kinetic characteristics of THC by tyrosinase. This procedure has the advantages of a short analysis time, straightforward measurement techniques and reproducibility. In addition, it also allows the study of tyrosinase inhibitors, such as tropolone.
Jiménez-Atiénzar
M
M
Departamento de Bioquímica y Biología Molecular A, Universidad de Murcia, Espinardo 30080, Murcia, Spain. mja@um.es
Pérez-Gilabert
M
M
Cabanes
J
J
Escribano
J
J
Gandía-Herrero
F
F
García-Carmona
F
F
D001572:Benzofurans / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D002599:Chalcone / Q000737:chemistry / Q000378:metabolism; D057075:Enzyme Assays; D006863:Hydrogen-Ion Concentration; D006900:Hydroxylation; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008956:Models, Chemical; D015394:Molecular Structure; D014442:Monophenol Monooxygenase / Q000378:metabolism*; D010084:Oxidation-Reduction; D013053:Spectrophotometry / Q000379:methods*
D001572:Benzofurans; C416232:aureusidin; D002599:Chalcone; D006899:Mixed Function Oxygenases; C416231:aureusidin synthase; D014442:Monophenol Monooxygenase