Journal of Biochemical and Biophysical Methods, 21 (1990) 35-46

35

Elsevier JBBM 00818

A new spectrophotometric assay for dopachrome tautomerase Pilar Aroca, Francisco Solano, Jos6 C. Garcia-Borr6n and Jos6 A. Lozano Departamento de Bioquimica y Biolog[a 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 ddpachrome oxidoreductase and dopachrome tautomerase have been proposed for the enzyme. So far, this enzyme has been assayed at 475 nm 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 before use, and (2) the rate of dopachrome decoloration in the absence of the enzyme is not negligible, and, furthermore, is enhanced by non-enzymatic agents. In order to overcome these problems, we present a new procedure that combines: (1) a quantitative, fast and easy way to prepare dopachrome from L-dopa by sodium periodate oxidation; (2) a spectrophotometric 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 dopachrome into 5,6-dihydroxyindole. 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 [1]. 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 Bioquimica y Biologla Molecular, Facultad de Medicina, Universidad de Murcia, 30100 Murcia, Spain. 0165-022X/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

36 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 m a n y years, tyrosmase 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 co catalyze the same reaction that occurs spontaneously in its absence, namely the decarboxylation of dopachrome to yield 5,6-dihydroxyindole ( D H I ) [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 ( D H I C A ) [8-10]. Thus. this reaction is m 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 p H also results in an absorbance decrease at this wavelength, although it involves a different mechanism. with loss of CO 2 to yield D H I [5,7,11]. Therefore. the absorbance decrease at 475 n m does not discriminate between the two colorless compounds. D H I and D H I C A . 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 m a y 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 determined discontinuously by H P L C separation of D H I and D H I C A 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, D H I C A and D H I even in acidic media [11], as well as the necessity of specialized equipment. It has been shown that dopachrome can be converted in either D H [ or D H I C A , depending on the p H of the reaction media [15,16]. Another important factor to be taken into account in the determination 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 mos~ commonly used method involves the oxidation by silver oxide at p H 6.8 followed by filtration [1,5-9,12 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]o In this paper, we present a new spectrophotometric assay in the ultraviolet region. Due to the auxochromic effect of the carboxyl group on the indole ring, the enzyme-catalyzed formation of D H I C A from dopachrome leads to an increase in

37 absorbance at 308 nm, while the spontaneous dopachrome evolution to D H I 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 g-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?O and sucrose were from Merck (Darmstadt, F.R.G.). The inorganic salts NaIO4, KIO3, ZnSO 4 • 7 H 2 0 , NiSO 4 • 7H20, C u S O 4 • 5 H 2 0 and CoC12 • 6 H 2 0 , were from Probus (Spain). Chelex 100 (sodium form) was purchased from Bio-Rad (Richmond, CA), DEAE-cellulose was from W h a t m a n (Kent, U.K.) and Ultrogel AcA-34 from LKB (Sweden). D H I C A was a kind gift from Dr. WyIer (Lausanne, Switzerland). D H I C A 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 Waters System, with a resistance of more than 10 M~2 • cm 1.

Animals and melanomas B16 mouse melanoma melanocytes were originally a kind gift from Dr. V. Hearing (NIH, Bethesda, U.S.A.). They had been maintained by serial transpIantation on hybrid mice obtained from male DBA and female C57,/B1 (Panlab, Spain). Only male mice at 6 - 8 weeks of age were used for tumor transplantation, and they were injected subcutaneously with approx. 105 viable cells. After 3 - 4 weeks, visible tumors were excised, some 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 0 C. Freshly excised tumors were washed twice in ice-cold 10 m M phosphate buffer, p H 6.8, containing 0.25 M sucrose and 0.1 m M EDTA. The washed tumors were weighed and homogenized in a Polytron homogenizer (power setting at 7), in the same buffer supplemented with 0.1 m M PMSF. The homogenate was centrifuged at 700 × g for 20 rain. The supel-natant 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 m M phosphate buffer, p H 6.8, containing 1% Brij 35. The suspension was incubated 30 rain at 4 ° C with gentle stirring,

38 and centrifuged at 105 000 × 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 a m m o n i u m sulfate and incubated overnight. After centrifugation at 11 000 × g for 30 rain, 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 m M phosphate buffer, p H 6.8, extensively dialyzed against this buffer, and further purified by hydroxyapatite batch chromatography [101. The purified fractions were concentrated in an Amicon Ultrafiltration Cell and applied to an Ultrogel AcA-34 column (52 × 2.6 cm), for gel filtration chromatography. The column was equilibrated and eluted with 50 m M phosphate buffer, p H 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,

Dopachromepreparation 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 m M sodium phosphate, p H 6.0, and the r e @ r e d 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 Ag20 [5,6,8,181 with or without treatment with Chelex 100 to remove traces of silver [131. D o p a c h r o m e preparations were used immediately. One unit of dopachrome tautomerase was defined as the amount of enzyme that catalyzes the transformation of I/~mol dopachrome per min at 30 o C. For the calculation of enzyme units, and in order to allow for a better comparison with the results reported by others, the amount of dopachrome transformed was estimated at 475 n m (e = 3700 M - 1 cm-1), unless otherwise stated.

Results and Discussion

Fig. 1 shows the initial A475 and the evolution with time of fresh dopachrome solutions prepared from 0.1 m M L-dopa either by oxidation with stoichiometric N a [ O 4 or with excess of Ag20, with optional Chelex 100 treatment to remove metal traces. Severn aspects should be pointed out: firstly, the L-dopa oxidation by periodate ( 1 : 2 stoichiometry) was fast and stoichiometric, as shown by the initial A475 obtained (e = 3700 M -1 cm-1). On the other hand, silver oxide treatment was not stoichiometric, as evidenced by the lower initial A475. 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 Ag20 m e t h o & !n this case, treatment with Chelex 100 [13]

39

j" 0.4-

z~

A47

0.2,

TJme(mio) Fig. 1. 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 (A) according to Ref. 13, or only filtered (zx)[5,6,8,18]. The dopachrome was kept in 10 mM phosphate buffer, pH 6.0, at 30 ° C in all cases. After 2 h, darkening was visible only in samples obtained with Ag20.

of freshly p r e p a r e d a n d filtered d o p a c h r o m e solutions i m p r o v e d their s t a b i l i t y as shown b y the d e l a y e d d a r k e n i n g of d o p a c h r o m e in c o m p a r i s o n to f i l t r a t i o n a l o n e [5,6,8[. However, a n d even when filtration a n d Chelex t r e a t m e n t were c o n s e c u t i v e l y carried out, the d a r k e n i n g of d o p a c h r o m e solutions was faster t h a n the o n e o b s e r v e d for p e r i o d a t e oxidized p r e p a r a t i o n s , as j u d g e d b y the e v o l u t i o n of the A475 (Fig. 1). Similar stability p r o b l e m s of A g 2 0 - p r e p a r e d d o p a c h r o m e solutions h a v e b e e n r e c e n t l y r e p o r t e d [18]. Finally, the c o p r o d u c t of the o x i d a t i o n o f L - d o p a b y p e r i o d a t e , iodate, d i d n o t affect the stability of d o p a c h r o m e w h e n it was a d d e d to d o p a c h r o m e solutions up to a 1 0 m M c o n c e n t r a t i o n (results n o t shown). M o r e o v e r , p r e i n c u b a t i o n of c r u d e enzyme s a m p l e s with either p e r i o d a t e or i o d a t e at c o n c e n t r a tions up to 1 m M did n o t affect the rate of the e n z y m e - c a t a l y z e d d e c o l o r a t i o n of dopachrome.

40

151

4



i Absorbance

300

400

500 Waveiengt h (nm)

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 rain (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, Universit6 de Lausanne, Lausanne, Switzerland) was indistinguishable from 4. Bearing these points in mind, d o p a c h r o m e was always prepared by stoichiometric oxidation with periodate. The spectrum of a 0.1 m M solution of freshly prepared d o p a c h r o m e in 10 m M phosphate buffer, p H 6.0, is shown in Fig. 2 (trace 1). As it is well k n o w n [1], two peaks can be seen, one in the visible region (Xmax = 475 nm, e = 3700 M -1 cm -1) and the other in the n e a r - U V region (Xm~x = 305 rim, s = 10 960 M -1 c m - 1 ) . D o p a c h r o m e d i s a p p e a r a n c e has been usually followed b y the absorbance decrease of the visible peak at 475 nm. However, it is k n o w n that the spontaneous d o p a c h r o m e decoloration at neutral p H proceeds with decarboxylation, while enzymatic decoloration proceeds b y a different mechanism: the e n z y m e catalyzes a tautomerization and prevents decarboxylation [8-10]. The spectra of d o p a c h r o m e solutions decolorized,in the presence (yielding mainly D H I C A ) or the absence (yielding D H I ) of d o p a c h r o m e tautomerase are also shown in Fig. 2 (traces 2 and 3). The visible absorbance at 475 n m is decreased in b o t h cases, but the absorbance changes in the U V region are dependent on the presence of the enzyme. Since d o p a c h r o m e tautomerase prevents C O 2 release, the carboxylic group remains on the indole ring at position 2. The resulting spectral changes are indicative of an auxochromic effect: a shift of the peak to greater wavelengths and an increase in the absorption coefficient (s). In the absence of the enzyme, d o p a c h r o m e undergoes a decarboxylation to yield D H I . Therefore, the spectra are characterized b y a shift of the U V peak to smaller wavelengths and a decrease in the absorptivity coefficient. D H I and, to a lesser extent, D H I C A , are unstable products that undergo new oxidations and polymerizations to yield a eumelanin polymer. Therefore, the "clean'

41

/

/

/

/

/

//

/

/

/

J

10

20 ~Ab475

2

4

Enzyme (mUnits)

Fig. 3. Linear correlation between the amount of enzyme in the reaction medium (munits), and the absolute value of the absorbance changes at 308 nm (11) and 475 nm (n), for a constant substrate concentration 0.1 mM dopachrome in 10 mM phosphate buffer, pH 6.0. Inset: Correlation of the observed absorbance changes at 308 and 475 nm.

spectra of these c o m p o u n d s is difficult to o b t a i n . However. Fig. 2 also shows the s p e c t r u m of a freshly p r e p a r e d solution of D H I C A (trace 4) o b t a i n e d as d e s c r i b e d b y I t o a n d W a k a m a t s u [19]. T h e s p e c t r u m was i d e n t i c a l for our p r e p a r a t i o n a n d for the s t a n d a r d o b t a i n e d f r o m Dr. Wyler. It c a n b e seen that the s p e c t r u m is quite similar to the one o b t a i n e d after a d d i n g d o p a c h r o m e t a u t o m e r a s e to a d o p a c h r o m e s o l u t i o n (trace 2). O n the other h a n d , our a t t e m p t s to synthesize D H I a c c o r d i n g to Ref. 19 were unsuccessful, a n d u n f o r t u n a t e l y , a s p e c t r u m of this c o m p o u n d c o u l d n o t b e o b t a i n e d . I n spite of that, the s p e c t r a 2 a n d 3 in Fig. 2 s h o u l d b e very close to the spectra of D H I C A a n d D H I , respectively. T h e s p e c t r o p h o t o m e t r i c d a t a of the stable 5,6-dimethoxy derivatives dissolved in e t h a n o l were Xmax = 319, e = 16 850 M -1 c m -1 for the 2 - c a r b o x y l a t e d i n d o l e a n d Xm~x = 295 nm, e = 7240 M -1 c m -1 for the d e c a r b o x y l a t e d unit [1]. F u r t h e r d a t a on the 5 , 6 - d i h y d r o x y i n d o l e s were r e p o r t e d after c h e m i c a l synthesis of D H I C A (Xma × - 320 rim, e = 16 850 M -1 c m - 1 ) a n d D H I (X .... = 302 nm, e = 6110 M -1 c m - 1 ) [20], b u t b o t h the p o s i t i o n a n d the a b s o r b a n c e of the m a x i m a are p H - d e p e n d e n t [11]. A c c o r d i n g to these data, the o p t i m a l w a v e l e n g t h to e s t i m a t e D H I C A a p p e a r a n c e , a n d h e n c e t a u t o m e r a s e activity, should b e a r o u n d 315 320 n m at p H 6.0. H o w e v e r , the analysis of the d a t a in o u r h a n d s i n d i c a t e d that 308 n m was the b e s t p o i n t to e s t i m a t e the initial r a t e of t a u t o m e r i z a t i o n , p r o b a b l y due to the coexistence of d o p a c h r o m e a n d D H I C A in the solution. Fig. 3 shows the linear relationship b e t w e e n the a m o u n t of p u r i f i e d t a u t o m e r a s e p r e s e n t in the r e a c t i o n m e d i u m a n d the a b s o l u t e value of the a b s o r b a n c e c h a n g e s at

42 475 n m a n d 308 rim. It can be seen that the absolute value of the a b s o r b a n c e changes per u m t time is higher at 308 n m t h a n at 475 nm. Moreover. a linear correlation of the a b s o r b a n c e changes at 308 a n d 475 n m can be observed (Fig. 3 inset), with a slope slightly higher t h a n 1. indicative of a higher sensitivity of the m e a s u r e m e n t at 308 rim. Such a n increased sensitivity at 308 n m is to be expected o n the basis of the spectral characteristics of dopachrome. D H I C A a n d D H I . W h e n the reaction rate was analyzed at different substrate c o n c e n t r a t i o n s , a n d using a c o n s t a n t a m o u n t of purified enzyme, similar results were o b t a i n e d at 475 a n d 308 nm, wRh a K m a r o u n d 50 ~ M for dopachrome. F o r routine m e a s u r e m e n t s , a d o p a c h r o m e c o n c e n t r a t i o n of 0.1 m M was used. Even though higher substrate c o n c e n t r a t i o n s could be desirable for some experiments: they result i n high b l a n k s b o t h at 475 a n d at 308 nmo The value 0.1 m M was therefore chosen as a compromise between s a t u r a t i o n of the enzyme a n d c o n v e n i e n t blanks. The sensitivity of the s p e c t r o p h o t o m e m c d e t e r m i n a t i o n is only slightly higher when measuring the increase in a b s o r b a n c e at 308 n m as opposed to the a b s o r b a n c e decrease at 475 rim. The m a j o r advantage of the m e a s u r e m e n t at 308 n m is the ~mproved specificity: d o p a c h r o m e conversion i n t o b o t h D H I a n d D H [ C A results i n a decrease in a b s o r b a n c e at 475 n m . b u t onty the enzyme-catalyzed d e c o l o r a t i o n of d o p a c h r o m e solutions leads to a n increase in the U V a b s o r b a n c e of the sample. I n our opinion, this feature is highly desirable, since d o p a c h r o m e is relatively u n s t a b l e a n d some agents usually present i n m e l a n o c v t e extracts can accelerate the rate of non-specific decoloration [21]o F o r instance, a high p r o t e i n c o n c e n t r a t i o n or ~races of metal ions wou]d simulate the d o p a c h r o m e t a u t o m e r a s e activity if the

1.2

~308

g|1 O.

"'

~3 BSA

0.8

u Ttme

4

(rnln)

Fig. 4. Effect of BSA concentration on the rate of dopachrome decomposition. Absorbance change at 308 nm of 0.1 mM solutions of dopachrome in 10 mM phosphate buffer, pH 6.0, and in the presence of 0 (1), 0.2 (2), 0.45 (3) and 0.9 rag/m1 (4) of BSA. Inserts: BSA concentration (mg/ml) and absorbance changes (expressed as enzyme activity, in mU/min) as a function of BSA concentration.

43

I

\

Absor~ance

300

400

500

wavelength (nrn)

Fig. 5. Effect of metal ions on the evolution with the time of 0.1 m M dopachrome solutions. T h e spectra were obtained in the absence of metal ions (1), or the presence of 0.1 m M Ni 2+ (2), Co 2+ (3), Cu 2+ (4) and Zn 2+ (5). The spectra were recorded after 30 rain incubation in the presence of the metal ion, except spectrum 4 which was recorded after 10 min. The spectrum in dashed line corresponds to a freshly prepared dopachrome sample.

absorbance decrease at 475 n m is followed, but not if the absorbance increase at 308 n m is recorded. The effect of protein (BSA) concentration on the rate of dopachrome decomposition can be followed at 475 or 308 nm. When the absorbance of the dopachrome solution is followed at 475 nm, a decrease in absorbance is observed, which is dependent on the BSA concentration (not shown). However, the absorbance at 308 nm also decreases (Fig. 4), thus showing that the BSA-dependent acceleration of dopachrome decomposition is not related to its tautomerization to DHICA. On the other hand, Fig. 5 shows the Vis-UV absorption spectra of the spontaneous dopachrome decomposition of a reaction mixture containing 100 # M dopachrome in the absence or the presence of different metal ions at a 0.1 m M final concentration. According to Palumbo et al. [13], Cu(II) markedly accelerated the rate of dopachrome disappearance, and the solution became dark quite rapidly, thus masking the evolution of dopachrome. For the other cations, different trends were observed at 475 and 308 nm. At 475 nm, the order of absorbance decrease rates was Ni(II) > C o ( I I ) > Z n ( I I ) > control. These results are totally consistent with those reported by Palumbo et al. [13]. At 308 rim, the order of absorbance decrease rates was Z n ( I I ) > C o ( I I ) > Control > Ni(II). These spectral changes might result from the coexistence of two reactions involving dopachrome and catalyzed by metal ions: one leading to D H I C A and the other one to D H I . 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 n m

44

allows for a discrimination between true dopachrome tautomerase activity 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 [9,131, exactly what the tautomerase does. A similar conclusion was reached by the discontinuous, HPLC method described by Leonard et aL ,[9]. Fortunately, the Ni(II) content of melanin-containing tissues is very iow.

0.45

0.25 0

T i m e (rain)

1.1

1

0..c

Tims(~in)

Fig. 6. Evolution with time of the absorbance at 475 nm (a) and 308 nm (b) of a 0.1 mM dopachrome solution in 10 mM phosphate buffer, pH 6.0, in the absence (1) or in the presence of 1.81 mU of crude enzyme in both assays (2).

45 The ability of the spectrophotometric measurement between tautomerase activity and dopachrome

at 308 n m t o d i s c r i m i n a t e

decomposition mediated by a high

p r o t e i n c o n t e n t , m e t a l i o n s o r o t h e r c o n t a m i n a n t s is i l l u s t r a t e d in t h e e x p e r i m e n t s u m m a r i z e d i n F i g . 6. w h e r e t h e r e a c t i o n r a t e w a s f o l l o w e d at 475 a n d 308 n m , u s i n g a c r u d e e n z y m e p r e p a r a t i o n . T h e n o n - s p e c i f i c d e c r e a s e in a b s o r b a n c e a t 475 nm

proceeded with

an

absorbance

i n c r e a s e at

308

magnitude. This change clearly shows that DHICA

nm

of the

same

order

of

is a c t u a l l y b e i n g f o r m e d , a

c o n c l u s i o n that c a n n o t be r e a c h e d u n e q u i v o c a l l y o n the basis of m e a s u r e m e n t s at 475 n m .

Simplified description of the modified method and its applications A new spectrophotometric method is presented to measure the dopachrome tantomerase acnvitv. This method consists m monitoring the absorbance increase at 308 nm of the following reaction mixture. For a 1 ml quartz spectrophotometer cuvette, the dopachrome solution is prepared 'in situ' by mixing within the cuvette 0.160 ml of a 0.6 mM solution of dopa in 10 mM phosphate, pH 6.0. with 0.04 ml of 4.8 mM NaIO 4 in the same buffer and buffer to 0.9 ml. The concentrations and volumes of the dopa and periodate solutions can be changed provided that the 1 : 2 stoichiometry is preserved and that a final concentration of dopacbxome close to 0.1 mM is obtained. 0.1 ml of enzyme solution is then added and the rate of absorbance increase at 308 nm at 30 ° C is followed in a thermostated spectrophotometer. ]7he solutions should be prewarmed at this temperature. Since the spontaneous dopachrome decompositiortis not negligible under these conditions and results in a decrease in absorbance, a blank should be performed by replacing the enzyme solution by the adequate amount of the corresponding buffer, and the absolute value of the observed change in absorbance should be added to the absorbance increase obtained for the enzyme. The advantages of this method are the absence of interferences with other agents that could simulate this enzymauc acuvi~y if absorbance at 475 nm is measured. Furthermore. the preparation of dopachrome in stoichiometric conditions avoids the presence of unoxidized •-dopa in the assay media, so that tautomerase activity can be measured even in the presence of tyrosinase.

Acknowledgements This work has been supported, by grants from DGCYT

tNo. PB87-0698) (Spain)

a n d f r o m t h e C o m u n i d a d A u t o n o m a d e M u r c i a . P. A r o c a , is r e c i p i e n t o f a fellowship f r o m the I n s t i t u t o de F o m e n t o de M u r c i a (Spain). T h e a u t h o r s w i s h to t h a n k D r . V. H e a r i n g f o r p r o v i d i n g t h e B 1 6 - F 1 0 m a l i g n a n t m e l a n o c y t e s a n d D r . H . W y l e r for D H I C A standard.

References 1 Mason, H.S. (1948) The chemistry of me!anins. J. Biol. Chem. 172, 83-99. 2 Garcia-Carmona, F., Garcia-Cfinovas, F. and Lozano, J.A. (1982) Kinetic study of the pathway of melanization between L-dopa and dopachrome. Biochim. Biophys. Acta 714, 124-131. 3 Hearing, V.J. and Jimenez, M. (1987) Mammalian tyrosinase. The critical regulatory control point in melanocyte pigmentation. Int. J. Biochem. 19, 1141-1147.

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A new spectrophotometric assay for dopachrome tautomerase.

The existence of a new enzyme involved in mammalian melanogenesis has been recently reported. The names dopachrome oxidoreductase and dopachrome tauto...
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