Tyrosinase activity assay

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). 

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. 

Mushroom tyrosinase was extracted as described by Ingebrigtsen and Flurkey (J. Food Sci., in press). Tyrosinase activity was monitored using either catechol, dopa or tyrosine as the substrates. All assays were carried out in the presence and absence of 0.1% SDS (w/v) to detect active and latent enzyme activities. The catechol oxidase activity of tyrosinase was assayed in 50 mM phosphate (pH 6.0) containing 10 mM catechol and the absorbance monitored at 410 nm (25-26). The dopa oxidase activity of tyrosinase was assayed in 50 mM phosphate (pH 6.0) containing 5 mM L-dopa and the absorbance monitored at 475 nm. The tyrosine hydroxylase activity of tyrosinase was assayed in 33 mM phosphate (pH 6.0) containing 0.33 mM L-tyrosine and the absorbance monitored at 280 nm. Protein content was determined by the method of Lowry et al. (26). 

Tyrosinase activity was monitored in four stages of mushroom development. Small pins (0-.5 cm), large pins (.5-1 cm), imnature and mature mushrooms were classified by cap size, gill development, and veil covering (K. Dahlberg, personal communication). These samples were assayed for active and latent tyrosinase activity after extraction in the absence of phenolic adsorbents (Fig. 2). 

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. 

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. 

Catechol as an Activator of Tyrosinase. The phenolase activity of tyrosinase has been studied less completely than the catecholase activity, partly because of the lack of a satisfactory assay procedure. The phenolase reaction, however, is characterized by a lag time which can be abolished by adding dihydroxyphenylalanine (DOPA), the immediate product of the hydroxylation reaction 29S4, 102, 117), This phenomenon has been described by several investigators (29-34) and is illustrated in Figure 12, from Pomerantz and Warner (117), using the enzyme from Hamster melanoma. The same phenomenon has been analyzed by Duckworth and Coleman (102) for the mushroom enzyme. In the absence of DOPA, maximum velocity of the hydroxylase reaction is not reached for several minutes. Pomerantz and Warner (117) devised a convenient assay for the phenolase reaction by determining the radio-... 

To create a fast and simple method for monitoring of human exposure to neuropathic OPs, a principal new approach to NTE activity analysis has been developed in joint study of the Institite of Physiologically Active Compounds Russian Acad. Sci. and Chemical Department of Moscow State University [88,91,92], Recently, a new biosensor for NTE assay was introduced using a tyrosinase carbon-paste electrode to detect phenol produced by the hydrolysis of phenyl valerate. In this type biosensor phenol is quantified by measuring electroreduction of the generated o-quinone on a graphite electrode (Fig. 6) [88,91 ]. The tyrosinase carbon-paste electrode improved the sensitivity of the NTE assay 10-fold compared to the colorimetric method or an earlier amperometric technique based on oxygen detection [92]. Moreover, the new electrode operates in a... 

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