A new spectrophotometric assay for dopachrome tautomerase

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names dopachrome oxidoreductase and dopachrome tautomerase have been proposed for the enzyme. So far, this enzyme has been assayed at 475 nrn on ...
Journal of Biochemical and Biophysical Methods, 21 (1990) 35-46 Elsevier

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JBBM 00818

A new spectrophotometric assay for dopachrome tautomerase Pilar Aroca, Francisco Solano, José C. García-Borrón and José A. Lozano Departamento de Bioquimica y Biología Molecular, Facultad de Medicina, Universidad de Murcia, Murcia, Spain

(Received 11 December 1989) (Accepted 15 February 1990)

Summary The existence of a new enzyme involved in mammalian melanogenesis has been recently reported. The names dopachrome oxidoreductase and dopachrome tautomerase have been proposed for the enzyme. So far, this enzyme has been assayed at 475 nrn on the basis of its ability to catalyze dopachrome decoloration. This method presents two major problems, derived from the instability of the substrate (dopachrome): (1) dopachrome must be prepared immediately befare use, and (2) the rate of dopachrorne decoloration in the absence of the enzyrne is not negligible, and, furthermore, is enhanced by non-enzymatic agents. In arder to overcome these problems, we present a new procedure that combines: (1) a quantitative, fast and easy way to prepare dopachrorne frorn L-dopa by sodiurn periodate oxidation; (2) a spectrophotornetric method in the UV region, at 308 nm, based on following the absorbance increase due to the enzyme-specific tautomerization of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid as opposed to the absorbance decrease due to the spontaneous decarboxylative transformation of dopa .. chrorne into 5,6-dihydroxyindo!e. The advantages of these methods as compared to the previously used procedures are discussed.

Introduction

Mammalian melanogenesis is a biosynthetic process that takes place in the specialized cells called melanocytes, and can be divided in two phases [l]. The first one, the oxidation of L-tyrosine to dopachrome, is catalyzed by the bifunctional enzyme tyrosinase (EC 1.14.18.1) [2,3]. The second one consists in the oxidation of dopachrome to yield melanin. This second phase of the process can occur spontaCorrespondence address: P. Aroca, Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Murcia, 30100 Murcia, Spain. 0165-022X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedica! Division)

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neously 'in vitro' through a number of intermediate metabolites such as 5,6-dihydroxyindoles, indolequinones and oligomers arising from these compounds [1,4]. Thus, for many years, tyrosinase has been considered the only enzyme involved in mammalian melanogenesis. Nevertheless, further studies suggested the existence of other factors controlling mammalian melanogenesis [5]. One of these factors was called dopachrome conversion factor, on the basis of its ability to catalyze dopachrome decoloration. Although the first properties reported for this factor appeared to exclude an enzymatic nature [6], the association of the activity to a protein factor was unequivocally established in 1984, and the name dopachrome oxidoreductase was proposed for the enzyme [7). The enzyme was thought to catalyze the same reaction that occurs spontaneously in its absence, namely the decarboxylation of dopachrome to yield 5,6-dihydroxyindole (DHI) [7]. However, more recent studies performed with either crude or highly purified preparations have shown that the action of dopachrome oxidoreductase leads to the non-decarboxylative rearrangement of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA) [8-10]. Thus, this reaction is in fact a keto-enolic tautomerization, and we have proposed the name dopachrome tautomerase (EC 5.3.2.3) for the enzyme [10). Enzymatic activity is usually measured spectrophotometrically by following the dopachrome consumption at 475 nm (e= 3700 M- 1 cm- 1 ) [5-8). However, the spontaneous decomposition of dopachrome at neutral pH also results in an absorbance decrease at this wavelength, although it involves a different mechanism, with loss of C0 2 to yield DHI [5,7,11]. Therefore, the absorbance decrease at 475 nm does not discrirninate between the two colorless compounds, DHI and DHICA. Moreover, several reagents, such as metal ions, have been shown to promote dopachrome decoloration [12-14]. Therefore, these agents accelerate the absorbance decrease at 475 nm, and may lead to serious artifacts, specially when the activity of crude extracts is measured. Other specific assays for the enzyme have been described. Dopachrome-converting activity can be deterrnined discontinuously by HPLC separation of DHI and DHICA in acidic media and quantitation of both products by fluorometric [9) or spectrophotometric detection [5,13]. The main problems of these methods are the tedious sample preparation and the relative instability of dopachrome, DHICA and DHI even in acidic media [11], as well as the necessity of specialized equipment. lt has been shown that dopachrome can be converted in either DHI or DHICA, depending on the pH of the reaction media [15,16). Another important factor to be taken into account in the deterrnination of this activity is the way in which dopachrome is prepared. Due to its relative instability, dopachrome should be prepared by L-dopa oxidation immediately before use. So far, the most commonly used method involves the oxidation by silver oxide at pH 6.8 followed by filtration [l,5-9,12-13). Under these conditions, the yield of L-dopa oxidation does not exceed 80%. Therefore a significant amount of unoxidized L-dopa is unavoidably present in the dopachrome solution [7,8,12]. In this paper, we present a new spectrophotometric assay in the ultraviolet region. Due to the auxochrornic effect of the carboxyl group on the índole ring, the enzyme-catalyzed formation of DHICA from dopachrome leads to an increase in

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absorbance at 308 nm, while the spontaneous dopachrome evolution to DHI leads to an absorbance decrease at this wavelength. The method allows for the discrimination of the true enzymatic activity as opposed to the non-enzymatic dopachrome decoloration promoted by other agents. Moreover, we propose the preparation of dopachrome by the stoichiometric oxidation of L-dopa by sodium periodate [17]. This procedure has two major advantages: unoxidized L-dopa is absent from the reaction medium, and dopachrome samples do not need to be filtered to eliminate the excess of insoluble oxidizer. The advantages of this preparation method are discussed.

Materials and Methods Reagents L-Dopa, BSA, EDTA, PMSF and Brij 35 were from Sigma Chemical Co. (St Louis, MO, U.S.A.). Sodium monobasic and dibasic phosphates, sodium hydroxide, Ag 20 and sucrose were from Merck (Darmstadt, F.R.G.). The inorganic salts Nal04 , KI03 , ZnS04 • 7H 20, NiS04 • 7H 20, CuS04 • 5H 20 and CoC1 2 • 6H 20, were from Probus (Spain). Chelex 100 (sodium form) was purchased from Bio-Rad (Richmond, CA), DEAE-cellulose was from Whatman (Kent, U.K.) and Ultrogel AcA-34 from LKB (Sweden). DHICA was a kind gift from Dr. Wyler (Lausanne, Switzerland). DHICA was also obtained in our laboratory as described elsewhere [19]. All reagents were of the highest purity commercially available and were used without further purification. All solutions were prepared using double-distilled water passed through a Milli-Q W aters System, with a resistance of more than 10 M.Q · cm- 1• Animals and melanomas Bl6 mouse melanoma melanocytes were originally a kind gift from Dr. V. Hearing (NIH, Bethesda, U.S.A.). They had been maintained by serial transplantation on hybrid mice obtained from male DBA and female C57 /Bl (Panlab, Spain). Only male mice at 6-8 weeks of age were used for tumor transplantation, and they were injected subcutaneously with approx. 10 5 viable cells. After 3-4 weeks, visible tumors were excised, sorne of them were used for new implantations and the others for enzymatic preparations. Purification of dopachrome tautomerase All steps were carried out at 0-4 ºC. Freshly excised tumors were washed twice in ice-cold 10 mM phosphate buffer, pH 6.8, containing 0.25 M sucrose and 0.1 mM EDTA. The washed tumors were weighed and homogenized in a Polytron homogenizer (power setting at 7), in the same buffer supplemented with 0.1 mM PMSF. The homogenate was centrifuged at 700 X g for 20 min. The supernatant was further centrifuged at 11 000 X g for 30 min in a Sorvall SS-34 rotor. The resulting melanosomal pellet was resuspended in 10 mM phosphate buffer, pH 6.8, containing 1 % Brij 35. The suspension was incubated 30 min at 4 ° C with gentle stirring,

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and centrifuged at 105 000 X g for 60 min, and the supernatant was used as a source of the enzyme. The melanosomal extract was then brought to 35% saturation with ammonium sulfate and incubated overnight. After centrifugation at 11 000 X g for 30 min, the supernatant was brought to 60% saturation and again centrifuged. The pellet was resuspended in a small volume of 0.1 % Brij 35 in 10 mM phosphate buffer, pH 6.8, extensively dialyzed against this buffer, and further purified by hydroxyapatite batch chromatography [10]. The purified fractions were concentrated in an Amicon Ultrafiltration Cell and applied to an Ultrogel AcA-34 column (52 X 2.6 cm), for gel filtration chromatography. The column was equilibrated and eluted with 50 mM phosphate buffer, pH 6.8, containing 0.1 % Brij 35, and the fractions were tested for enzymatic activity. The fractions with the highest specific activity were pooled and used for these studies. Dopachrome preparation Because of its instability, dopachrome was chemically prepared 'in situ' by L-dopa oxidation. Since the chemical oxidation of L-dopa by periodate leads to its quantitative conversion into dopachrome [17], fresh solutions of dopachrome were prepared by mixing a solution of L-dopa in 10 mM sodium phosphate, pH 6.0, and the required volume of a solution of sodium periodate so as to achieve a 1 : 2 molar ratio of L-dopa/ periodate. Alternatively, dopachrome was sometimes prepared using Ag 2 0 [5,6,8,18] with or without treatment with Chelex 100 to remove traces of silver [13]. Dopachrome preparations were used immediately. One unit of dopachrome tautomerase was defined as the amount of enzyme that catalyzes the transformation of 1 µmol dopachrome per min at 30 ° C. For the calculation of enzyme units, and in arder to allow for a better comparison with the results reported by others, the amount of dopachrome transformed was estimated at 475 nm (e= 3700 M- 1 cm- 1 ), unless otherwise stated.

Results and Discussion

Fig. 1 shows the initial A 475 and the evolution with time of fresh dopachrome solutions prepared from 0.1 mM L-dopa either by oxidation with stoichiometric Nal04 or with excess of Ag 20, with optional Chelex 100 treatment to remove metal traces. Several aspects should be pointed out: firstly, the L-dopa oxidation by periodate (1: 2 stoichiometry) was fast and stoichiometric, as shown by the initial A 475 obtained (e = 3700 M- 1 cm - l ). On the other hand, sil ver oxide treatment was not stoichiometric, as evidenced by the lower initial A 475 • Furthermore, it was time-consuming, since filtration and Chelex 100 treatments are needed to eliminate the excess of silver. Secondly, and since the oxidation by periodate is quantitative, no L-dopa is left in solution. Since L-dopa is a substrate for tyrosinase, the absence of L-dopa is a major advantage for the assay of dopachrome tautomerase in the presence of tyrosinase. Thirdly, the periodate method yielded a dopachrome solution more stable than the Ag 2 0 method. In this case, treatment with Chelex 100 [13]

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0.4

0.2

60

20

Time(min)

Fig. l. Absorbance changes of fresh dopachrome preparations. Dopachrome was formed by oxidation of 0.1 mM L-dopa using periodate (•) or silver oxide. In this last case, freshly prepared dopachrome solutions were filtered and treated with Chelex 100 (•) according to Ref. 13, or only filtered (L>) [5,6,8,18]. The dopachrome was kept in 10 mM phosphate buffer, pH 6.0, at 30 ° C in ali cases. After 2 h, darkening was visible only in samples obtained with Ag 2 0.

of freshly prepared and filtered dopachrome solutions improved their stability as shown by the delayed darkening of dopachrome in comparison to filtration alone (5,6,8]. However, and even when filtration and Chelex treatment were consecutively carried out, the darkening of dopachrome solutions was faster than the one observed for periodate oxidized preparations, as judged by the evolution of the A 475 (Fig. 1). Similar stability problems of Agp-prepared dopachrome solutions have been recently reported (18]. Finally, the coproduct of the oxidation of L-dopa by periodate, iodate, did not affect the stability of dopachrome when it was added to dopachrome solutions up to a 10 mM concentration (results not shown). Moreover, preincubation of crude enzyme samples with either periodate or iodate at concentrations up to 1 mM did not affect the rate of the enzyme-catalyzed decoloration of dopachrome.

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1.5

Absorbance

Wavelength 1nm1

Fig. 2. UV-Vis spectra of newly formed dopachrome (1), and evolution of this compound in the presence of enzyme (1.6 mU) after 30 min (2) or in the absence of the enzyme after 3 h (3). The reaction mixtures consisted of 0.1 mM dopachrome in 10 mM phosphate buffer, pH 6.0. The assays proceeded at 30 ° C. The spectrum 4 corresponds to a 0.1 mM solution of DHICA obtained according to Ref. 19. The spectrum of a standard DHICA provided by Dr. Wyler (Institut de Chimie organique, Université de Lausanne, Lausanne, Switzerland) was indistinguishable from 4.

Bearing these points in rnind, dopachrome was always prepared by stoichiometric oxidation with periodate. The spectrum of a 0.1 mM solution of freshly prepared dopachrome in 10 mM phosphate buffer, pH 6.0, is shown in Fig. 2 (trace 1). As it is well known [l], two peaks can be seen, one in the visible region O'max = 475 nm, e = 3700 M- 1cm - l) and the other in the near-UV region ( ;\ max = 305 nm, e = 10 960 M- 1 cm- 1 ). Dopachrome disappearance has been usually followed by the absorbance decrease of the visible peak at 475 nm. However, it is known that the spontaneous dopachrome decoloration at neutral pH proceeds with decarboxylation, while enzymatic decoloration proceeds by a different mechanism: the enzyme catalyzes a tautomerization and prevents decarboxylation [8-10]. The spectra of dopachrome solutions decolorized in the presence (yielding mainly DHICA) or the absence (yielding DHI) of dopachrome tautomerase are also shown in Fig. 2 (traces 2 and 3). The visible absorbance at 475 nm is decreased in both cases, but the absorbance changes in the UV region are dependent on the presence of the enzyme. Since dopachrome tautomerase prevents C02 release, the carboxylic group remains on the índole ring at position 2. The resulting spectral changes are indicative of an auxochrornic effect: a shift of the peak to greater wavelengths and an increase in the absorption coefficient (e). In the absence of the enzyme, dopachrome undergoes a decarboxylation to yield DHI. Therefore, the spectra are characterized by a shift of the UV peak to smaller wavelengths and a decrease in the absorptivity coefficient. DHI and, to a lesser extent, DHICA, are unstable products that undergo new oxidations and polymerizations to yield a eumelanin polymer. Therefore, the 'clean'

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D Control > Ni(II). These spectral changes might result from the coexistence of two reactions involving dopachrome and catalyzed by metal ions: one leading to DHICA and the other one to DHI. These reactions might proceed at different rates depending on the concentration and nature of the metal ions [13,14]. In any case, it can be concluded that the spectrophotometric method at 308 nm

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allows for a discrimination between true dopachrome tautomerase act1v1ty and metal-catalyzed dopachrome decomposition. Among the metal ions tested, only Ni(II) could simulate enzymatic activity, due to the ability of this cation to prevent dopachrome decarboxylation and to catalyze DHICA formation [9,13], exactly what the tautomerase