Tyrosinase concentration measurement

Two substrates are required in the tyrosinase-catalyzed reaction, phenolic substrate (dopa) and dioxygen. The conditions described in the experiment are such that the reaction mixtures are saturated with dissolved dioxygen. Therefore, when measurements are made for Ku, only the concentration of dopa is limiting, so the rate of the reaction depends on dopa concentration. The dopachrome assay is extremely flexible, as it can be applied to a variety of studies of tyrosinase. 

Whether an inhibitor acts in a competitive or noncompetitive manner is deduced from a Lineweaver-Burk or direct linear plot using varying concentrations of inhibitor and substrate. In separate assays, two substances will be added to the dopa-tyrosinase reaction mixture, and the effect on enzyme activity will be quantified. The structures of the potential inhibitors, cinnamic acid and thiourea, are shown in Figure E5.9. The inhibition assays must be done immediately following the KM studies. To measure inhibition, reaction rates both with and without inhibitor must be used and the tyrosinase activity must not be significantly different. If it is necessary to do the inhibition studies later, the Ku assay for L-dopa must be repeated with freshly prepared tyrosinase solution. 

If the tyrosinase preparation is homogeneous, the concentration of enzyme may be determined by absorbance measurement at 280 nm. The absorption... 

The enzyme/substrate (tyrosinase/wool hydrolysate) mixture (20 pL) was diluted widi 980 pL of distilled water, 70 pL of ethylenediamine, and 50 pL of 2M etfaylenediainine dihydrochloride (pH 11). The mixture was incubated at 50°C for 2 h in die dark, and then the fluorescence intensity was measured using a UVA IS spectrofluorom o (Tecan). The excitation and emission wavelengdis were at 420 and 543 nm, respectively. The concentrations of the DOPA residue in the preparations were estimated using a standard fluorescence curve of DOPA [8]. For the Dopa Quinone (DQ) quantification M6TH (3-methyl-2-benzothiazolinone hydrazone hydrochlcnide monohydrate) was used. MBTH reacts with DQ to form a pink pigmoit widi Xnm at 505 nm. The assay solution was prepared by mixing 480 pL of the en me/substrate reaction mixture, 980 pL of 4.3% (v/v) DMF (dimethyl formamide) in distiUed water, and 580 pL of 20.7 mM MBTH. The total volume was 2 mL. Tbe reacticm mixture was incubated at 25 C for 10 min before the absorbance measurement (at 505 nm) [8]. 

Tyrosinase is inhibited by various substances, among which are aromatic carboxylic acids. The inhibitory character of these compounds is linked to the presence of the benzene ring [26]. Inhibition by members of benzoic and cinammic acid series was studied in the literature [27-31]. It is known that benzoates and cinnamates are naturally found in red wines with high concentrations. So during the analysis performed for the determination of amount of phenolic compounds, we should consider the effect of these substances on the measurements. 

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