Tyrosinase assays

Steiner, U., Schhemann, W., and Strack, D., Assay for tyrosine hydroxylation activity of tyrosinase from betalain-forming plants and cell cultures, Anal. Biochem., 238, 72, 1996. 

Tyrosinase, a copper-containing oxidoreductase, catalyzes the orthohydroxy-lation of monophenols and the aerobic oxidation of catechols. The enzyme activity will be assayed by monitoring the oxidation of 3,4-dihydroxyphenyl-alanine (dopa) to the red-colored dopachrome. The kinetic parameters Ku and Vmax will be evaluated using Lineweaver-Burk or direct linear plots. Inhibition of tyrosinase by thiourea and cinnamate will also be studied. Two stereoisomers, L-dopa and D-dopa, will be tested and compared as substrates. 

Several kinetic characteristics of mushroom tyrosinase will be examined in this experiment. A spectrophotometric assay of tyrosinase activity will be introduced and applied to the evaluation of substrate specificity, Ku of the natural substrate, 3,4-dihydroxyphenylalanine (L-dopa), and inhibition characteristics. 

Many assays for tyrosinase activity have been developed. Procedures in the literature include use of the oxygen electrode, oxidation of tyrosine followed at 280 nm, and oxidation of dopa followed at 475 nm. The most convenient assay involves following the tyrosinase-catalyzed oxidation of dopa by monitoring the initial rate of formation of dopachrome at 475 nm (Figure E5.8). 

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. 

Part B Tyrosinase level for kinetic assay—1 hour. 

Before kinetic constants can be evaluated, it is critical to find the correct concentration of enzyme to use for the assays. If too little enzyme is used, the overall absorbance change for a reaction time period will be so small that it is difficult to detect differences due to substrate concentration changes or inhibitor action. On the other hand, too much enzyme will allow the reaction to proceed too rapidly, and the leveling off of the time course curve as shown in Figure E5.7 will occur very early because of the rapid disappearance of substrate. A rate that is intermediate between these two extremes is best. For the dopachrome assay, it is desirable to use the level of tyrosinase that gives a linear absorbance change at 475 nm for 2 minutes. 

Now that the appropriate enzyme level has been determined, the kinetic constants may be evaluated. The Ku for L-dopa can be obtained by setting up the same assay as in part B, except that the factor to vary will be the concentration of L-dopa. The concentration of L-dopa in part B was sufficient to saturate all the tyrosinase active sites, so the rate depended only on the enzyme concentration. In part C, L-dopa levels will be varied over a range that is nonsaturating. 

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. 

To set up the inhibition assay, prepare a table similar to Table E5. 1. Inhibitor should appear in the list of reagents before tyrosinase. Use the same level of tyrosinase and the same dopa stereoisomer as in part C. Vary the amount of dopa as in part C. A constant amount of inhibitor (cinnamic acid or thiourea) should be added to each cuvette. You will have to determine this level of inhibitor by trial and error. The desired inhibition rate with saturating substrate is about 50% of the uninhibited rate. Add all reagents except tyrosinase, mix well, and determine the blank rate, if any. Add tyrosinase, mix, and immediately record AA75 for 2 minutes. From recorder traces or graphs of A475 vs. time, calculate AA/min for each assay. 

Add another column to your table, label it jttmoles product formed per minute, and calculate the appropriate rate for each enzyme concentration. Prepare a graph of rate (ju,mole/min on the ordinate, y axis) vs. enzyme concentration in each assay (mg, on the abscissa, x axis). Connect as many of the points as possible with a straight line passing through the origin. If most of the points are on this line, the assay and the standard curve can be used to quantify an unknown level of tyrosinase. The standard curve also provides the experimenter with a choice of enzyme levels to use for further kinetic studies. 

I. Behbahani, S. Miller, and D. O Keeffe, Microchem.J. 47,251-260 (1993). A Comparison of Mushroom Tyrosinase Dopaquinone and Dopachrome Assays. ... 

Figure 42. 8-Anilino-l-naphthalene sulfonic acid (ANS) binding assay of the protyrosinase (pro-TY, —) and acid-activated tyrosinase (acid T Y, —).

Li et al. (1990) developed an assay to measure the diphenol oxidase activity of tyrosine by following the conversion of 3,4-dihydroxymandelic acid (DHMA) to 3,4-dihydroxybenzaldehyde (DHBZ). Tyrosinase is involved in the formation of melanotic pigments in a wide variety of plants and animals. 

The assay was used to measure the activity of a commercial preparation of mushroom tyrosinase, and the activity in cell-free hemolymph from mosquitoes. 

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