2,3,4-trihydroxyacetophenone (2,3,4-thap) as a substrate for mushroom tyrosinase

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2,3,4-TRIHYDROXYACETOPHENONE (2,3,4-THAP) AS A SUBSTRATE FOR MUSHROOM TYROSINASE' VARDA KAHN Department of Food Science, Agricultural Research Organization The Volcani Center, P.O. Box 6, Bet Dagan, Israel Received for Publication October 6, 1989 Accepted for Publication January 22, 1990 ABSTRACT 2,3,4-Trihydroxyacetophenone (2,3,4-THAP) can serve as a substrate for mushroom tyrosinase with a K, value of 1.2 mM. The product(s) formed is yellow, characterized by a peak at 420430 nm. A lag period in 2,3,4-THAP oxidation to the yellow product(s) in the presence of ascorbate indicates that the initial product(s) is an o-quinone of 2,3,4-THAP. An oxime, characterized by a broad peak at 510-650 nm, is the likely product formed between o-quinone of 2,3,4-THAP and NH,OH. The E, of the o-quinone of 2,3,4-THAP was estimated to be 1.6 X lo" M-' cm-' at 425 nm. During relatively long incubation periods, the peak of the yellow product(s) shifrsfrom 420425 nm to 430440 nm; the solution remains yellow and trans- parent for at least a week and no precipitate isformed. TheJinalyellowproduct(s) is probably a low molecular weight polymer of THAP-o-quinone (dimer, tetra- mer, etc). INTRODUCTION Trihydroxyphenones such as 2,4,5 -trihydrox ybutyrophenone (2,4,5-THBP) are antioxidants that are used to inhibit oxidation processes in fats, paraffin waxes, mineral oil, carotene extracts, ready-mixes of poultry feeds and cattle fodder (Emanuel and Lyaskovska 1967). In the course of our studies on the oxidation of the antioxidant 2,4,5-THBP by mushroom tyrosinase (unpublished data), we tested the ability of related antioxidants to serve as a substrate for the enzyme. The data presented in this paper provide evidence that 2,3,4-trihydroxyacetophenone (2,3,4-THAP) can serve as a substrate for mushroom tyrosinase. 'Contribution No. 2521-E. from the Agricultural 1989 series. Research Organization, The Volcani Center, Bet Dagan, Israel. Journal of Food Biochemistry 14 (1990) 189-198. All Rights Resenvd. 0 Copyright 1990 by Food & Nutrition Press, (nc., Trumbull, Connecticut. 189

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2,3,4-TRIHYDROXYACETOPHENONE (2,3,4-THAP) AS A SUBSTRATE FOR MUSHROOM TYROSINASE'

VARDA KAHN

Department of Food Science, Agricultural Research Organization The Volcani Center, P . O . Box 6, Bet Dagan, Israel

Received for Publication October 6, 1989 Accepted for Publication January 22, 1990

ABSTRACT

2,3,4-Trihydroxyacetophenone (2,3,4-THAP) can serve as a substrate for mushroom tyrosinase with a K , value of 1.2 mM. The product(s) formed is yellow, characterized by a peak at 420430 nm.

A lag period in 2,3,4-THAP oxidation to the yellow product(s) in the presence of ascorbate indicates that the initial product(s) is an o-quinone of 2,3,4-THAP. An oxime, characterized by a broad peak at 510-650 nm, is the likely product formed between o-quinone of 2,3,4-THAP and NH,OH. The E, of the o-quinone of 2,3,4-THAP was estimated to be 1.6 X lo" M-' cm-' at 425 nm.

During relatively long incubation periods, the peak of the yellow product(s) shifrs from 420425 nm to 430440 nm; the solution remains yellow and trans- parent for at least a week and no precipitate is formed. TheJinal yellowproduct(s) is probably a low molecular weight polymer of THAP-o-quinone (dimer, tetra- mer, etc).

INTRODUCTION

Trihydroxyphenones such as 2,4,5 -trihydrox ybutyrophenone (2,4,5 -THBP) are antioxidants that are used to inhibit oxidation processes in fats, paraffin waxes, mineral oil, carotene extracts, ready-mixes of poultry feeds and cattle fodder (Emanuel and Lyaskovska 1967).

In the course of our studies on the oxidation of the antioxidant 2,4,5-THBP by mushroom tyrosinase (unpublished data), we tested the ability of related antioxidants to serve as a substrate for the enzyme. The data presented in this paper provide evidence that 2,3,4-trihydroxyacetophenone (2,3,4-THAP) can serve as a substrate for mushroom tyrosinase.

'Contribution No. 2521-E.

from the Agricultural 1989 series.

Research Organization, The Volcani Center, Bet Dagan, Israel.

Journal of Food Biochemistry 14 (1990) 189-198. All Rights Resenvd. 0 Copyright 1990 by Food & Nutrition Press, (nc., Trumbull, Connecticut. 189

190 VARDA KAHN

EXPERIMENTAL

Materials

Mushroom tyrosinase (grade Ill), 2,3,4-THAP, 2,4,5-THBP, L-ascorbic acid, NH,OH and phenylhydrazine were from Sigma. All other chemicals were reagent grade.

Assays

The rate of 2,3,4-THAP oxidation by mushroom tyrosinase was measured in a total volume of 3 mL containing 2,3,4-THAP, 47 mM sodium phosphate buffer (pH 6.5) and mushroom tyrosinase (added last) in the absence or presence of different additives as specified in the figure legends.

The rate of 2,3,4-THAP oxidation was followed at 425 nm. Activity (AOD 425 n d m i n ) was estimated from the initial linear portion of the absorbance versus time curves obtained. Spectral changes (350-600 nm) were traced at the rate of 100 n d m i n . Spectrophotometric measurements were carried out at 22- 24°C using a Varian DMS 90 spectrophotometer equipped with a recorder.

RESULTS

2,3,4-THAP in water is colorless but when it is incubated with mushroom tyrosinase in 47 mM sodium phosphate buffer (pH 6.5), a yellow product(s) forms immediately.

Rate of Enzymatic Oxidation of 2,3,4-THAP

Changes with time in the visible spectrum of the product(s) formed when 3.3 mM 2,3,4-THAP was incubated with 66.7 p,g/mL mushroom tyrosinase are shown in Fig. 1. The visible spectrum of the yellow product(s) formed during short incubation periods (1, 4, 8 min) was characterized by a peak at 420425 nm and a low trough at 380 nm. After relatively long incubation periods (i.e., 81 min), the peak shifted to 4 3 M 4 0 nm and the trough shifted to 395 nm and had much higher absorbance than that after a short incubation period (Fig. 1).

Incubation of 0.33 mM or 0.66 mM 2,3,4-THAP with 16.7 p,g/mL mushroom tyrosinase gave changes in the visible spectrum of the product(s) similar to those shown in Fig. 1 (data not presented).

Effect of Enzyme Concentration on Rate of Oxidation of 2,3,4-THAP

The rates of oxidation of 0.66 mM 2,3,4-THAP by various concentrations of mushroom tyrosinase were linear initially and then plateaued (Fig. 2A). Activities

( 2 , 3. 4,-THAD) AS A SUBSTRATE

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A (nm) FIG. 1, CHANGES WITH TIME IN THE V1SIBL.E SPECTRUM OF THE PRODUCT(S)

The reaction mixture included, in a total volume of 3 mL, 3.3 mM 2,3,4-THAP, 47 mM NaPi buffer (pH 6.5) and 200 pg mushroom tyrosinase (added last). A 0.3 mL aliquot of the sample was mixed with 0.9 mL water and the spectrum was scanned at the indicated times.

FORMED WHEN 2,3,4-THAP IS OXIDIZED BY MUSHROOM TYROSINASE

(AOD 425 nm/min) toward 0.66 d 2,3,4-THAP were linearly related to the amount of mushroom tyrosinase up to approximately 66.7 pgimL, respectively (Fig. 2B).

Effect of Substrate Concentration on Rate of Oxidation of 2,3,4-THAP: K, Determination

Rates of oxidation of 0.05 to 6 . 7 mM 2,3,4-THAP to the initial yellow product(s) (A,,,,, 425 nm) by 16.7 @mL mushroom tyrosinase were determined.

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Tyrosinase (pg I3mL) -

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Time (sec)

Tyrosinase ( pg 13 mL 1 FIG. 2. OXIDATION OF 2,3,4-THAP BY VARIOUS CONCENTRATIONS OF

MUSHROOM TYROSINASE The reaction mixture included, in a total volume of 3 mL, 0.66 mM 2,3,4-THAP, 47 mM NaPi buffer (pH 6.5), and various concentrations of mushroom tyrosinase (added last) as indicated. Activities (AOD 425 ndmin) , estimated from the initial linear portions of the kinetic curves of Part A, are plotted in part B as a function of various concentrations of mushroom tyrosinase.

( 2 , 3, 4,-THAD) AS A SUBSTRATE 193

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2,3,4-THAP ( mM ) FIG. 3. EFFECT OF SUBSTRATE CONCENTRATION ON THE RATE OF OXIDATION OF

The reaction mixture included, in a total volume of 3 mL: various concentrations of 2,3,4-THAP, 47 mM NaPi buffer (pH 6.5) and 16.7 kg/mL mushroom tyrosinase (added last). The rate of 2,3,4- THAP oxidation to the initial yellow product(s) was followed as a function of time. Velocity (activity) (AOD 425 nndmin) was estimated from the initial portions of the kinetic curves obtained. Lineweaver- Burk plot is shown in the inset.

2,3,4-THAP; K, DETERMINATION

Lineweaver-Burk plots (Webb 1963; Fig. 3:) gave a K, value for 2,3,4-THAP of 1.2 mM.

Effect of Ascorbate on Rate of Substrate Oxidation by Enzyme

Ascorbate is known to be an effective reductant of o-quinones. The rate of oxidation of 0.66 mM 2,3,4-THAP by 13.3 Fg/mL mushroom tyrosinase in the absence of ascorbate was linear for about 90 s, whereas a lag period was seen when 0.066-0.2 mM ascorbate was included in the reaction mixture; the higher the concentration of ascorbate, the longer was the lag period (Fig. 4).

Occurrence of the lag period in Fig. 4 indicates that the initial yellow product(s) formed when 2,3,4-THAP is oxidated by mushroom tyrosinase is a quinone that is reduced by ascorbate back to colorless 2,3,4-THAP.

194 VARDA KAHN

2.0 c Ascorbate (mM)

Time (min 1 FIG. 4. EFFECT OF VARIOUS CONCENTRATIONS OF ASCORBATE ON THE RATE OF

The reaction mixture included, in a total volume of 3 mL, 0.66 mM 2,3,4-THAP, 47 mM NaPi buffer (pH 6 . 5 ) , 13.3 p,g/mL mushroom tyrosinase (added last) and ascorbate as indicated.

2,4,5-THAP OXIDATION BY MUSHROOM TYROSINASE

Effect of NH,OH on Spectral Changes when 2,3,4-THAP is Oxidized by Enzyme

NH,OH can interact with quinones or with ketones to form oximes (Finley 1974). Interaction of NH,OH with quinones yields colored oximes because of benzene conjugation, while interaction of NH,OH with alkyl ketones yields colorless oximes. When 3.3 mM 2,3,4-THAP was oxidized by 16.7 p&mL mushroom tyrosinase in the presence of 13.3 mM NH,OH, the product(s) seen initially (1 min) was yellow and was characterized by a peak at 420-430 nm (Fig. 5A). During further incubation periods (3, 8, 17, 25 min) a shoulder appeared at 500-650 nm and the product(s) was violet. The absorbancy of the shoulder at 500-650 nm increased with time, while that at 420-430 decreased with time (Fig. 5A). By comparison, under identical conditions but in the absence of NH,OH, the sample remained yellow (420-430 nm) even after 25 min in- cubation and a shoulder at 500-650 nm was not observed (Fig. 5B).

The isosbestic point at 480 nm (Fig. 5A) shows that the yellow product(s) formed initially is converted to the violet product(s) as a function of time. This is further evidence that the yellow product(s) formed initially are quinones of 2,3,4-THAP which interact with NH,OH to yield violet oximes.

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em of Product@) Formed when 2,3,4-THAP is Oxidized by Enzyme

The apparent molar extinction coefficient (E,) of the quinones formed when 2,3,4-THAP was oxidized by mushroom tyrosinase was estimated by the Waite method (1976). The rate of oxidation of 16.6, 33.2, 49.8 and 66.4 pA4 2,3,4- THAP (monitored at 425 nm) by 133 pg/mL mushroom tyrosinase is shown in Fig. 6. Maximum absorbance (at 425 nm) in the plateau phase of the hyperbolic curves obtained in Fig. 6 was plotted in the inset as a function of 2,3,4-THAP concentration. From the linear relationship between these two parameters an apparent molar extinction coefficient at 425 nm of the initial oxidation products of 2,3,4-THAP (2,3,4-THAP-quinone) was estimated to be 1.6 x lo4 M-' cm-I.

DISCUSSION

Oxidation of 2,3,4-THAP by mushroom tyrosinase gave a yellow product(s) characterized by a peak at 420-430 nm. The K, value of 2,3,4-THAP for

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FIG. 6. MOLAR EXTINCTION COEFFICIENT (e,) OF THE INITIAL YELLOW PRODUCT@) FORMED WHEN 2,3,4-THAP IS OXIDIZED BY ENZYME

The reaction mixture included, in a total volume of 3 mL; 2,3,4-THAP (in M as indicated), 47 mM NaPi buffer (pH 6.5) and 133 p,g/mL mushroom tyrosinase (added last). Maximum OD (425 nm) reached at the plateau phase was plotted in the inset as a function of 2,3,4- THAP concentration.

(2, 3, 4;THAD) AS A SUBSTRATE 197

mushroom tyrosinase was 1.2 mM, which is higher than that for 4-methyl catechol (0.08 mM) or DL-DOPA (0.48 mM) (Kahn and Miller 1987).

Observation that the oxidation of 2,3,4-THAP by mushroom tyrosinase in the presence of ascorbate (Fig. 4) is characterized by an initial lag period suggests that the initial yellow oxidation product(s) is a quinone. The quinones can interact with NH,OH to form a violet product(s), absorbing at 480-650 nm (Fig. 5A); probably each forms a mono oxime and a dioxime (Finley 1974).

The quinones formed initially polymerize with time to low m.w. polymers as judged by the observation that the initial peak. at 4 2 M 2 5 nm shifted to a longer wavelength (430-440 nm) following relatively long incubation periods (Fig. 1).

A reaction mixture containing up to 6.7 mM 2,3,4-THAP and 100 Fg/mL mushroom tyrosinase, in the presence of sodium phosphate buffer (pH 6.5), remained yellow and transparent for at least one week. The absence of a pre- cipitate indicates that, under these conditions, the final yellow product(s) is not a high m.w. polymer. (It was not possible to determine the potential polymer- ization of higher concentrations of 2,3,4-THAP-quinone to a high m.w. polymer because of solubility limitations of 2,3,4-THAP).

The molar extinction coefficient (1.6 x LO M - ' cm-' at 425 nm) of the initial oxidation product(s) of 2,3,4-THAP is very high relative to the initial oxidation product(s) of 4-methyl catechol and 4-tert-butyl catechol, and high relative to the initial oxidation products of DL-DOPA and dopamine (Waite 1976). The high molar extinction coefficient of the initial oxidation products of 2,3,4-THAP makes it a useful substrate for assaying relatively low levels of o-dihydroxyphenolase activities of tyrosinase.

ABBREVIATIONS

2,3,4-THAP = 2,3,4-trihydroxyacetophenone. 2,4,5-THBP = 2,4,5-trihy- droxybutyrophenone. Mushroom tyrosinase, monophenol mono-oxygenase (monophenol dihydroxyphenylalanine: oxygen oxidoreductase, E.C. 1.14.18.1); also known as phenolase, catecholase, polyphenoloxidase (PPO) and cresolase.

ACKNOWLEDGMENTS

This research was supported by grant No. 1-1074-1986 from the United States- Israel Binational Agricultural Research and Development Fund (BARD). The excellent technical assistance of Ms. Varda Zakin is gratefully acknowledged.

REFERENCES

EMANUEL, N. M. and LYASKOVSKA, 'Y. N. 1967. The Inhibition of Fat Oxidation Processes, Pergamon Press, New York.

198 VARDA KAHN

FINLEY, K. T. 1974. The addition and substitution chemistry of quinones. In The Chemistry of the Quinoid Compounds, Part 2, (S. Patai, ed.) pp. 877- 1144, John Wiley & Sons, New York.

KAHN, V. and MILLER, R. W. 1987. Tiron as a substrate for mushroom tyrosinase. Phytochemistry 26, 2459-2466.

TRAUTNER, E. M. and ROBERTS, A. H. 1950. The chemical mechanism of the oxidative deamination of amino acids by catechol and polyphenolase. Aust. J . Sci. Res. Ser. B. 3 , 356380.

WAITE, J. H. 1976. Calculating extinction coefficients for enzymatically pro- duced o-quinones. Anal. Biochem. 75, 21 1-218.

WEBB, J. L. 1963. In Enzyme and Metabolic Inhibitors, Vol. 1, p. 149, Ac- ademic Press, New York.