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Chemical intermediates in dopamine oxidation by tyrosinase, and kinetic studies of the process
ARCHIVES
OF BIOCHEMISTRY
AND
Vol. 235, No. 2, December,
BIOPHYSICS
pp. 438-448, 1984
Chemical Intermediates in Dopamine Oxidation by Tyrosinase, and Kinetic Studies of the Process M. JIMENEZ,
Departamentos
F. GARCIA-CARMONA, J. A. LOZANO, de Bioquimica
F. GARCIA-CANOVAS, F. MARTINEZ*
y de *Q&mica-Fisica,
Received February
J. L. IBORRA,’
AND
Universidad
de Murcia,
Mu&a,
Spain
14, 1984, and in revised form June 5, 1984
A minor pathway for dopamine oxidation to dopaminochrome, by tyrosinase, is proposed. Characterization of intermediates in this oxidative reaction and stoichiometric determination have both been undertaken. After oxidizing dopamine with mushroom tyrosinase or sodium periodate in a pH range from 6.0 to 7.0, it was spectrophotometrically possible to detect o-dopaminoquinone-H+ as the first intermediate in this pathway. The steps for dopamine transformation to dopaminochrome are as follows: dopamine - o-dopaminequinone-H+ - o-dopaminequinone - leukodopaminochrome - dopaminochrome. No participation of oxygen was detected in the conversion of leukodopaminochrome to dopaminochrome. Scanning spectroscopy and graphical analysis of the obtained spectra also verified that dopaminequinone-H+ was transformed into aminochrome in a constant ratio. The stoichiometry equation for - dopamine + dopaminochrome. The this conversion is 2 o-dopaminequinone-Hf pathway for dopamine oxidation to dopaminochrome by tyrosinase has been studied as a system of various chemical reactions coupled to an enzymatic reaction. A theoretical and experimental kinetic approach is proposed for such a system; this type of mechanism has been named “Enzymatic-chemical-chemical” (E&C). Rate constants for the implied chemical steps at different pH and temperature values have been evaluated from the measurement of the lag period arising from the accumulation of dopaminochrome that took place when dopamine was oxidized at acid pH. The thermodynamic activation parameters of the chemical steps, the deprotonation of dopaminequinone-H+ to dopaminequinone, and the internal cyclization of dopamineCC:19x4 Academic press, I,,~. quinone to leukodopaminochrome have been calculated.
It has been established that the two major catabolic pathways of catecholamines are o-methylation and oxidative deamination (1). Occurrence of a minor route in which the dihydric phenolic moieties are oxidized to the corresponding oquinones, and the side chains cyclized to form trihydroxyindole derivatives, is a further possibility. Catalysis of the latter type of reaction by various biological tissues and fluid preparations, by certain 1 To whom correspondence should be addressed, at Departamento Interfacultativo de Bioquimica, Facultad de Medicina, Espinardo, Murcia, Spain. 0003-9861/84 $3.00 Copyright All rights
0 1984 by Academic Press, Inc. of reproduction in any form reserved.
438
oxidative enzymes, and by metalloproteins, has been demonstrated. However, there is disagreement whether the expected products of oxidation are present or involved in physiological processes, as well as whether the reaction is part of a normal or an abnormal metabolic pathway (2). In general, a third minor route has been considered with increasing interest during recent years due to the detection of the antitumoral effect of catecholamines (3), and of dopamine in particular (4). This antitumoral effect for malignant melanoma, in vitro as well as in vivo, has been ascribed to the reactivity of quinones pro-
KINETIC
STUDY
OF DOPAMINE
duced in the oxidation of catecholamines with tyrosinase (3-6). This enzyme was detected in large amounts in malignant melanoma cells (7-9). Oxidative processes of catecholamines by peroxidase (10, 11) or ceruloplasmine (12) via free radical production have been widely studied. However, oxidation by tyrosinase has not been analyzed. Tyrosinase oxidizes diphenols without the formation of free radicals (13,14). The direct product of this enzyme is, therefore, o-quinone, thought to undergo a series of chemical reactions following Raper-Mason’s classic scheme of melanization (15, 16). Tyrosinases from various sources have several properties in common, but also exhibit striking differences; for example, catechol is an excellent substrate for mushroom tyrosinase, but catechol derivatives with substituents in position 4, such as dopamine, noradrenaline, and adrenaline, have much lower oxidation rates. On the other hand, in the case of mammalian tyrosinase, unsubstituted catechol is a poor substrate, and catechol derivatives with the same substituents undergo oxidation at much greater rates (17). The present paper deals with the dopamine oxidation by sodium periodate and mushroom tyrosinase. Spectral characteristics of the oxidation products and their stoichiometry up to dopaminochrome have been established. The kinetic analysis of this pathway has also been studied as an enzymatic-chemical-chemical (E,CC) mechanism, such as described in a previous paper (18). Rate constants for each of the implied chemical steps have been evaluated, and the existence of o-dopaminequinone-H+ as the first intermediate in the enzymatic action is suggested. MATERIALS
AND
METHODS
Mushroom tyrosinase (monophenol monooxygenase, EC 1.14.18.1, 24 DOPA units/mg) and dopamine chlorhydrate were purchased from Sigma. Sodium metaperiodate was analytical grade from Merck. Spectra were recorded with an Aminco DW-2 spectrophotometer with scanning speeds up to 20 rim/s. Reference cuvettes contained, in all cases, all the components except substrate.
OXIDATION
BY TYROSINASE
439
Spectrophotometric measurements were carried out with an Aminco DW-2 spectrophotometer equipped with a Hewlett-Packard recorder with kinetic response, thus allowing the dead recording time to be minimized. When the rate constant of the chemical reaction was greater than 0.1 s-i, an Aminco-Morrow stopped flow accessory equipped with a Dasar TM unit was also used. Dopaminochrome accumulation was spectrophotometrically followed at 480 nm; at this wavelength neither dopamine nor any other possible intermediate present in the reaction medium shows absorbance. The reaction medium was 10 mM phosphate buffer (pH as indicated in each case), 2 mM dopamine (i.e., in saturated conditions), and the amount of enzyme necessary to reach b (& is the time needed to attain the 0.99 Vo/2 rate). In these conditions, absorbance increase was less than 0.2. Determination of intermediate dopaminoquinone-H+ with an absorption maximum at 390 nm was made under the same conditions as described above for dopaminochrome determination. Oxygen consumption was followed by a Rank oxygen electrode (Rank Brothers). Temperature was controlled using a Gilson bath and a Cole-Palmer digital thermistor with a SR f O.l”C. Protein concentration was determined by the Lowry method (19). RESULTS
In an attempt to identify the intermediate compounds generated by dopamine oxidation into dopaminochrome, spectrophotometric determinations were carried out upon oxidation of dopamine by tyrosinase and sodium periodate, respectively. Both compounds oxidize o-diphenols to the respective o-quinones.
Dopamine Oxidation by Tyrosinase Figure IA shows the visible spectra of the products formed in the dopamine oxidation by tyrosinase (12.5 pg/ml) at pH 6.1. Two absorbance maxima (390 and 480 nm, respectively) were detected at pH 6.1, simultaneously developing as the reaction progressed. The graphical analysis of the recordings of Fig. 1A by the matrix method of Coleman et al. (20) corresponded for three absorbing species in solution, as illustrated in Fig. 2A (in Miniprint). When oxidation was carried out at pH 7.0, only the maximum at 480 nm was observed. This suggested that a reaction product
440
JIMENEZ
ET AL.
0.6 A 03
350
450
Akim)
550
L 350
I 150
A(nml
I 550
FIG. 1. (A) Spectrophotometric recordings (from 350 to 550 nm) for the oxidation of dopamine by tyrosinase at 10°C in 10 mM sodium phosphate buffer, pH 6.1. Dopamine (2 UIM) was oxidized with tyrosinase (12.5 pg/mI). Scan speed was 20 rim/s. The first recording was started at 20 s from the beginning of the reaction. (B) Spectrophotometric recordings for the oxidation of dopamine by tyrosinase, with oxygen depletion, from 350 to 550 nm, at 10°C in 10 mM sodium phosphate buffer, pH 6.1. Dopamine (2 mM) was oxidized with tyrosinase (50 fig/ml). Scanning conditions were the same as expressed in (A). (C) Oxygen consumption corresponding to the reaction shown in (B).
was detectable, not at pH 7, but at pH 6.1. This product developed before the maximum at 480 occurred, and agreed with the X,,, established for o-quinone (21). On the other hand, the maximum at 480 nm corresponded to aminochrome (22). It can therefore be assumed that o-quinone is the direct product of the enzymatic action. When dopamine oxidation was achieved by mushroom tyrosinase at pH 6.1, with a large enzyme concentration, it was possible to obtain oxygen depletion in the assay medium within a few minutes of the start of the reaction (Fig. 1C). Figure 1B shows the visible spectrum for this oxidation, undertaken with four times the enzyme concentration used in Fig. IA. As can be seen, the initial recordings were similar to those obtained in Fig. 1A but, from recording No 4, an isosbestic point was defined at 418 nm; this suggested that the enzymatic reaction had stopped due to oxygen depletion. From tracing 4 onward, the transformation of accumulated dopaminequinone-H+ into its products could thus be seen. The graphical analysis for these last recordings (Fig. 2B, in Miniprint) shows that there are two kinetically related species.
Dopamine Oxidation by Periodate It is widely established that o-diphenol chemical oxidation by sodium periodate leads to o-quinone production (22). Figure 3A shows the visible spectrum for the dopamine chemical oxidation, at pH 6.1, with an excess of periodate ([NaIO$[dopamine] = 12.5). The maximum at 390 nm appeared immediately at the beginning of the reaction and then decreased; the maximum at 480 nm subsequently evolved. The appearance of an isosbectic point in these spectra (X = 403 nm) seemed to suggest that there was a transformation of dopaminequinone into dopaminochrome at a constant ratio. This can be confirmed by the matrix method of Coleman et al. (20) (Fig 4A in Miniprint). Figure 3B shows the visible spectrum for dopamine oxidation with insufficient sodium periodate ([NaIOJ[dopamine] = 0.25) at pH 6.1 and, once again, an initial maximum at 390 nm evolved toward another maximum at 480 nm. A new isosbestic point appeared at X = 423 nm. Graphical analysis confirmed that o-dopaminequinone was converted into dopaminochrome at a constant ratio (Fig. 4B, in Miniprint).
KINETIC
STUDY
L50
X(nm)
OF DOPAMINE
OXIDATION
I 350
550
350
“O
BY TYROSINASE
I L50
441
Xtnm)
I 550
hlnml
FIG. 3. Spectrophotometric recordings (from 350 to 550 nm) for the oxidation of dopamine by sodium periodate at 10°C in 10 mM sodium phosphate buffer, pH 6.1. Scan speed was up to 20 rim/s. the first scan was started at 40 s from the beginning of the reaction. (A) Dopamine (0.4 mM) was oxidized with 2.5 mM NaIO,. (B) Dopamine (2 mM) was oxidized with 0.5 mM NaIOl. (C) Dopamine (0.2 mM) was oxidized with 0.4 mM NaIO,.
When oxidation was carried out, with an equimolar ratio, until an absorbance increase could no longer be detected (Fig. 5), the existence of two compounds, aminochrome with two absorbance maxima (300 and 480 nm), and dopamine, with a maximum at 280 nm, was evident. This suggested that dopamine was present in the reaction medium. The addition of a new equivalent of periodate to the reaction mixture increased the value of absorbance at both 300 and 480 nm twofold. Two equivalents of periodate were therefore necessary to render an equivalent of dopaminochrome. These results, together with those shown in preceding figures, suggested a
set of steps as shown in Fig. 6, agreeing with the recently postulated pathway for L-DOPA oxidation by tyrosinase (23). Stoichiometry of Dopamine by Tyrosinase
Oxidation
To establish the stoichiometry of the dopamine oxidation processes, an assay was performed by following the absorbance increase at pH 6.5 and 15°C (Fig. 7) with the reaction time at 390 and 480 nm, respectively. The rate of enzymatic reaction was evaluated as the tangent, at t = 0, to the recording of absorbance at 390 nm, (curve B), and also as the slope of the linear zone of the dopaminochrome
JIMENEZ
442
ET AL.
0.17
A
0.085
Xhm) FIG. 5. Oxidation of dopamine by sodium periodate at 15’C in 10 mM sodium phosphate buffer, pH 6.5. Dopamine (0.4 mM) was reacted with 0.4 mM (A) and 0.8 mM (B) NaIO,.
accumulation curve (curve A). The o-quinone production rate was twofold greater than the dopaminochrome accumulation rate, when dopaminochrome was accumulated at constant rate. These results agree with the postulated mechanism for the steps that follow the enzymatic reaction (Fig. 6). Kinetic
Approach
According to the postulated pathway previously described, dopamine oxidation into dopaminochrome by tyrosinase can
T~rosinase
H
0
I
FIG. 6. Pathway proposed dopamine to dopaminochrome nase.
for the oxidation of catalyzed by tyrosi-
FIG. 7. Product formation (with time) for 2 mM dopamine oxidation with tyrosinase (10 pg/ml) at 15”C, pH 6.5. (A) Absorbance increase of dopaminochrome accumulation followed at 480 nm. (B) Absorbance increase of dopaminoquinone followed at 390 nm.
be considered as a mechanism of two chemical reactions coupled to the enzymatic step (mechanism E,CC); note the recycling, by a set of chemical reactions, of a product of the enzymatic reaction into a substrate of this same step, according to the expressed scheme in Fig. 7 and developed theoretically in the Appendix. Evaluation of the Apparent Rate Constant Dopamine oxidation to dopaminochrome by tyrosinase presented a marked lag period, as has previously been shown (Fig. ‘7). This lag period was independent of enzyme concentration, in accordance with the theoretical approach developed for the scheme of chemical reactions coupled to an enzymatic step (Mechanism E,CC), with the particular characteristics of the proposed pathway for this oxidative process. On the other hand, dopaminochrome accumulation must reach a steady-state condition, where the accumulation is half the enzymatic velocity for the dopamine conversion into dopaminoquinone (Eq. [lo] of Appendix). Dopaminochrome accumulation recordings at different enzyme concentrations are presented in Fig. 8. The resulting curves tend toward straight
KINETIC
0
60
STUDY
t Is)
OF DOPAMINE
OXIDATION
BY TYROSINASE
443
120
FIG. 8. (A) Dopaminochrome accumulation with time at pH 6.2 and 15°C followed by absorbance increase at 480 nm for different enzyme concentrations (rg/ml): (a) 2.5, (b) 4, (c) 8, and (d) 10. (B) Plot of dopaminochrome accumulation rate, under steady-state conditions, against enzyme concentration.
lines, with slopes directly related to the enzyme concentration (Fig. 8B), and their lag periods (defined as the intercept on the abscissa axis of the tangent, at t co, to the dopaminochrome accumulation curve), were independent of enzyme concentration (Fig. 8A), and can be assimilated to l/k,, (eq. [ll] of Appendix); therefore, kapp = 0.042 s-‘. This constant can also be obtained from the analysis of the accumulation curves with the reaction time (Eq. [12] of Appendix). Figure 9 shows an analysis for the curves of Fig. 8 according to that equation. From the slope of these lines an apparent constant with a significantly similar value (0.04 s-l) to the calculated value from the lag period has been evaluated; thus, kap values will always be calculated from the lag period measurements.
Variation of the Lag Period with pH and Temperature: Evaluation of the Specific Rate Constants The lag period presented a significant variation with pH and temperature. As can be observed in Table I, the lag period decreased for pH and temperature values between 6 and 7, and 10 and 35”C, respec-
FIG. 9. Semilogarithmic representation of (PI - [D&J against time, according to Eq. [12]. The values of [D] correspond to those of Fig. 1A.
tively, when the pH and temperature were increased. If the kinetic treatment, obtained by approximation to equilibrium, was acceptable for this system, a linear relation must exist between the lag period (twofold) and proton concentration, at a fixed temperature. From the linear regression-fitted plots, kc can be evaluated from the slope, and kl from the intercept on the ordinate axis (Fig. 10). The firstorder rate constants for deprotonation of dopaminoquinone-H+ to dopaminoquinone and for the cyclization of dopaminoquinone to leukodopaminochrome at different temperatures were expressed in Table II.
TABLE
I
RECIPROCALS OF THE FIRST-ORDER RATE CONSTANTS FOR THE CONVERSION OF DOPAMINOQUINONE-H+ INTO DOPAMINOCHROME AT SEVERAL PH AND TEMPERATURE VALUES l/k, pH/t (“C) 6.1 6.4 6.7 7.0
10
15
44.5 31.5 16.7 11.5
31.5 17.8 12.5 7.2
20 16
10 6.5 3.7
(s) 25 10 5.7 3.7 2.2
30 6 3.8 2.2 -
35 3.6 2.1 1.25 -
444
JIMENEZ
ET AL. 1
7
100 2 k&is)
K,b) 5-1 50
\1 0.1
0.4 [H'] @I
3.6
"*
FIG. 10. Plot of the reciprocals of observed rate constants against proton concentrations at different temperatures, in the process of conversion of dopaminequinone-H+ to dopaminochrome (see Eq. [ll]).
FIG. 11. Arrhenius plot for k, and kl, values of k, and kl correspond to those of Table II.
The value of the second ionization constant for dopamine [pK, = 10.6 (24)] should be preliminarily considered as the ionization constant for dopaminequinone-H+. The results obtained correspond closely to the theoretical solutions used to resolve the set of chemical reactions coupled to the enzymatic oxidation of dopamine by tyrosinase.
zation processes, Arrhenius plots were made using the k,- and kl-obtained values, as shown in Fig. 11. The calculated activation parameters for the cyclization process, as well as for deprotonation, are summarized in Table III; the high value for AS of the cyclization process can be attributed to a very favorable probability factor that should lead to a very fast cyclization.
Evaluaticm of Themnodynamic Parameters for the Deprotonation and Cgclization Processes For the evaluation of the activation energies for the deprotonation and cycli-
DISCUSSION
o-Dopaminequinone has been detected as the first product of tyrosinase action on dopamine, at pH conditions where the quinone was stabilized by the protonation of its amine group. This group acts as a nucleophillic group during the intramoTABLE II lecular reaction of cyclization to leukoaminochrome (addition Michael 1, 4). DoFIRST-ORDER RATE CONSTANTS (kI) FOR DEPROTONAis more stable than doTION OF DOPAMINOQUINONE-H+ TO DOPAMINOQUINONE paminequinone pH because it AND FOR THE CYCLIZATION OF DOPAMINOQUINONE TO paquinone at physiological has a greater value of pK, for its amine LEUKODOPAMINOCHROME AT VARIOUS TEMPERATURES group [dopamine, 10.2; dopa, 8.72 (24)]. kl This means that there is a smaller ratio (sf). r”c, (0 for deprotonated dopamine. The internal cyclization reaction is therefore less fa10 0.06 417 vored. These results agree with the greater 15 0.11 588 cytotoxicity for the dopamine tyrosinase 20 0.19 1162 system (5). 25 0.39 1811 On the other hand, o-quinone stability 30 0.46 3187 depends on pH. At pH 6.1, the quinonic 35 0.84 5312 intermediate was accumulated to a level “Derived from Eq. [ll] by using a value of KS detectable by graphical analysis (Fig. 1; = 2.51. lo-“. and Fig. 2 in Miniprint). The system
KINETIC
STUDY
OF DOPAMINE
OXIDATION
TABLE
E Deprotonation Cyclization
mol-‘)
445
III
THERMODYNAMIC ACTIVATION PARMETERS FOR THE DEPROTONATION DOPAMINOQUINONE AND FOR THE CYCLIZATION OF DOPAMINOQUINONE
(kcal
BY TYROSINASE
OF DOPAMINOQUINONE-Hi INTO INTO LEUKODOPAMINOCHROME
MS (kcal
mol-‘)
A’3 (kcal
mol-‘)
As* WJ)
18.3
17.7
18
-1
18.5
17.9
13
16.4
reaches the steady-state and dopaminochrome is accumulated, after a lag, at constant rate (curve A of Fig. 7). From the experiments of dopamine oxidation by sodium periodate (Fig. 5), two equivalents of periodate were considered necessary for the complete oxidation of one equivalent of dopamine to dopaminochrome. In the experiments undertaken with tyrosinase (Fig. ‘7), the rate of dopaminochrome accumulation was half that of o-dopaminoquinone-Hf. Both experiments allowed us to conclude that the stoichiometric equation for the conversion of o-quinone-H+ into aminochrome is 2 odopaminoquinone-H+ - dopamine + dopaminochrome. When dopamine oxidation was carried out with insufficient sodium periodate (Fig. 3B), an isosbestic point at 423 nm could be seen. Analysis of the spectra showed that there were two species (Fig. 4B of Miniprint). However, there are four kinetically related species: dopamine, o-dopaminequinone-H+, leukodopaminochrome, and dopaminochrome. This result can be explained by considering that the leukodopaminochrome concentration was too small to be detected by matrix analysis. This coincides with the great difference of oxidation-reduction potentials between the o-quinone/o-diphenol and halochrome/leukohalochrome pairs (25). Furthermore it should be taken into account that, during the whole reaction, 1 mol dopamine and 1 mol dopaminochrome originate at a constant ratio from 2 mol dopaminequinone-H+. A similar situation occurs in dopamine oxidation with very high concentrations of tyrosinase. In these conditions, the oxygen was depleted in the medium; from tracing No.
4 in Fig. lB, the test for two species was positive, and was assumed to be o-dopaminequinone-H+ and dopamine plus dopaminochrome (Fig. 2B of Miniprint). The experiments performed with sodium periodate in excess (Fig. 3A) showed an isosbestic point, different from the preceding one, at 403 nm, which can be explained by the transformation of dopaminequinone-H’ into dopaminochrome with a 1:l stoichiometry, since the excess sodium periodate prevents the accumulation of dopamine in the medium. Graphical analysis for two species was therefore applicable, odopaminequinone-H+ and dopaminochrome being identified. When dopamine oxidation by periodate was carried out under stoichiometric conditions, the cyclization and oxidation reactions took place simultaneously, so no isosbestic point occurred (Fig. 3C) and graphical analysis determined three species which were considered to be dopamine, o-dopaminequinone-H+, and dopaminochrome. An identical result was obtained with low amounts of tyrosinase, when the oxygen in the solution was not depleted (Fig. 1A; and Fig. 2A of Miniprint). A pathway, the postulated steps of which are different from those proposed by Lsvstad (26), can therefore be proposed (Fig. 7), since Lsvstad assumed the appearance of free radicals in dopamine oxidation by ceruloplasmine. Kinetic studies also confirmed that dopamine oxidation to dopaminochrome followed the same pathway previously proposed by us for LDOPA (18), and also established that odopaminequinone-H+ was more stable than o-dopaquinone-H+ at physiologic PH (5).
446
JIMENEZ APPENDIX
The postulated be schematically
ET AL.
and
mechanism in Fig. 6 can written as follows:
[31 By using the fractional concentration tor of Cha (28), fB, as,
kl
PI fB = [BH] + [B] = where A = dopamine, BH = dopaminequinone-H+, B = dopaminequinone, C = leukodopaminochrome, and D = dopaminochrome. The equation for dopaminochrome accumulation curve can be obtained if it is assumed that (i) the enzymatic reaction rate remains constant (V,), since the variation of dopamine concentration during the reaction time is very small. (ii) The constant (K) for the equilibrium BH + C P A + D is very high, in accordance with the electrochemical data (5). It can thus be seen that the leukodopaminochrome concentration in the reaction medium tends toward zero throughout the whole reaction. Therefore, the apparent rate constant of leukodopaminochrome decomposition is the same as that of formation (kJB]) at all times. (iii) The rate constants for the steps BH P B - C are high, as they correspond to the deprotonation and cyclization processes. It can thus be assumed that they are in pseudo steady state. A simplified model can therefore be formally written as shown in the following scheme, in agreement with the kinetical schemes resolution, where one part is in steady state and the other in presteady state conditions (27).
kpJH+] + kl + k, ’ ‘41
the differential equations model can be written as 2
2kc fB.
In view of the preceding considerations, we can write: = kl[BH] - (kJH+]
+ kJ[B] = 0, [l]
where [X] = [BH] + [B] + [C] li [BH] + [B]
PI
and
Integrating these equations, and taking into account Eq. [4] and that [X] = 0 at t = 0, the following equation
v, “I
= (2k,kl)/(k-,[H+]
+ kl + kc) %k.
X
1 -
e
k-dH+l+k,+S
PI
t >
can be established, where the apparent rate constant is defined as
2We Kap = k-,[H+] + kl + kc ’ rD1 = :
it + (2k,kc)/(k-,[A+] X e- k-iH+l+h+k
PI
+ kl + kc)
t 1
+ kl + kc)>
PI
for t - co, it is possible to define how D is accumulated when the system reaches its steady state,
A&X
F
of simplified
= v, - ZfBkdx]
- (2klk,)/(k_JH+] -A
fac-
[2]
ID”
= :
tt - (2k,%)/(ke,[;+]
+ kl + kc>*
[W The lag period (L) can be defined as the intercept on the abscissa of the straight zone prolongation of the D accumulation curve,
KINETIC
STUDY
OF DOPAMINE
2L~[H’l+L+L2 K&c k1 kc ka, ’
WI
where K,=$. 1
A plot of 2L against [H+] gives a straight line, the slope of which gives the value of kc, if K, is known, and the intercept on the ordinate axis the kl values. On the other hand, using Eqs. [g] and [9], it can be established that
WDI - PISS)= In
-
V,WdH+l+ h + kc) 2kch 2Ul t. [12]
k-,[H+] + kl + kc Hence a plot time gives a which enables rate constant
of ln([D] - [D],,) against straight line,-the slope of us to evaluate the apparent (kap).
ACKNOWLEDGMENTS This research has been partially supported by a grant from the Comision Asesora de Investigation Cientifica y Tecnica. M. Jimenez had a fellowship from Ministerio de Education y Ciencia. REFERENCES 1. MOLINOFF, P. B., AND AXELROD, J. (1971) Annu. Rev. Biochem 40, 465-500. 2. HARRISON, W. H. (1963) Arch. Biochem. Biophys. 101, 116-130. 3. WICK, M. M. (1980) Cancer Res. 40, 1414-1418. 4. WICK, M. M. (1979) J. Natl Cancer Inst. 63,14651467. 5. WICK, M. M. (1979) Cancer Treat. Rep. 63, 991997. 6. WICK, M. M. (1978) J Invest. DermatoL 71. 163164.
OXIDATION
BY TYROSINASE
447
7. POMERANTZ, S. H., AND LI, J. P.-C. (1973) Yale J. Biol Med 46, 541-552. 8. NISHIOKA, K. (1978) Eur. .I Biochem 85, 137146. 9. BURNETT, J. B. (1971) J. Bid Chem 246, 30793091. 10. VAN WOERT, M. H., AND AMBANI, L. M. (1974) A&v. Neural. 5, 215-223. 11. I&STAD, R. A. (1979) Gen Pkarmaco~ 10, 437440. 12. WALLAS, E., AND WALLAS, 0. (1961) Arch Biochem Biophys. 95,151-162. 13. SEIJI, M., SASAKI, M., AND TOMITA, Y. (1978) Tohoku K. Exp. Med. 125,233-245. 14. TOMITA, Y., SEIJI, M., AND IGARASHI, A. (1980) J. Invest. Dermatol 7. 377-380. 15. RAPER, H. S. (1928) Physiol. Rev. 8. 245-282. 16. MASON, H. S. (1948) J. Biol. Chem. 172, 83-99. 17. YASUNOBU, K. T. (1953) in Pigment Cell Biology (Gordon, M., ed.), p. 583, Academic Press, New York. 18. GARCIA-CARMONA, F., GARCIA-CANOVAS, F., IBORRA, J. L., AND LOZANO, J. A. (1982) B&him Bie phys. Acta 717, 124-131. 19. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. BioL Chem. 193. 265-275. 20. COLEMAN, J. S., VARGA, L. P., AND MASTIN, S. H. (1970) Iwg. Chem 9. 1015-1020. 21. YASUNOBU, K. T., PETERSON, E. W., AND MASON, S. H. (1959) J. Biol. Chem 234, 3291-3295. 22. GRAHAM, D. G., AND JEFFS, P. W. (1977) J. BioL Chem. 252, 5729-5734. 23. GARCIA-CANOVAS, F., GARCIA-CAF~MONA,F., IBORFLA, J. L., AND LOZANO, J. A. (1982) J. Biol Chem. 257, 8738-8744. 24. GRAY, D. O., AND WEITZMAN, P. D. J. (1968) in Data for Biochemical Research (Dawson, R. M., Elliot, D. C., Elliot, W. H., and Jones, K. M., eds.), pp. 22-23, Oxford Univ. Press (Clarendon), London/New York. 25. SWAN, G. A. (1974) Forts&. Chem Org. Naturst. 31, 521-582. 26. L&STAD, R. A. (1971) Acta Chem. Stand 25, 3144-3150. 27. RICARD, J., But, J., AND MEUNIER, J. C. (1977) Eur. J. Biochem. 80,581-592. 28. CHA, S. (1968) J. BioL Chem 243, 820-825.
448
JIMENEZ
ET AL.
OF BIOCHEMISTRY
AND
Vol. 235, No. 2, December,
BIOPHYSICS
pp. 438-448, 1984
Chemical Intermediates in Dopamine Oxidation by Tyrosinase, and Kinetic Studies of the Process M. JIMENEZ,
Departamentos
F. GARCIA-CARMONA, J. A. LOZANO, de Bioquimica
F. GARCIA-CANOVAS, F. MARTINEZ*
y de *Q&mica-Fisica,
Received February
J. L. IBORRA,’
AND
Universidad
de Murcia,
Mu&a,
Spain
14, 1984, and in revised form June 5, 1984
A minor pathway for dopamine oxidation to dopaminochrome, by tyrosinase, is proposed. Characterization of intermediates in this oxidative reaction and stoichiometric determination have both been undertaken. After oxidizing dopamine with mushroom tyrosinase or sodium periodate in a pH range from 6.0 to 7.0, it was spectrophotometrically possible to detect o-dopaminoquinone-H+ as the first intermediate in this pathway. The steps for dopamine transformation to dopaminochrome are as follows: dopamine - o-dopaminequinone-H+ - o-dopaminequinone - leukodopaminochrome - dopaminochrome. No participation of oxygen was detected in the conversion of leukodopaminochrome to dopaminochrome. Scanning spectroscopy and graphical analysis of the obtained spectra also verified that dopaminequinone-H+ was transformed into aminochrome in a constant ratio. The stoichiometry equation for - dopamine + dopaminochrome. The this conversion is 2 o-dopaminequinone-Hf pathway for dopamine oxidation to dopaminochrome by tyrosinase has been studied as a system of various chemical reactions coupled to an enzymatic reaction. A theoretical and experimental kinetic approach is proposed for such a system; this type of mechanism has been named “Enzymatic-chemical-chemical” (E&C). Rate constants for the implied chemical steps at different pH and temperature values have been evaluated from the measurement of the lag period arising from the accumulation of dopaminochrome that took place when dopamine was oxidized at acid pH. The thermodynamic activation parameters of the chemical steps, the deprotonation of dopaminequinone-H+ to dopaminequinone, and the internal cyclization of dopamineCC:19x4 Academic press, I,,~. quinone to leukodopaminochrome have been calculated.
It has been established that the two major catabolic pathways of catecholamines are o-methylation and oxidative deamination (1). Occurrence of a minor route in which the dihydric phenolic moieties are oxidized to the corresponding oquinones, and the side chains cyclized to form trihydroxyindole derivatives, is a further possibility. Catalysis of the latter type of reaction by various biological tissues and fluid preparations, by certain 1 To whom correspondence should be addressed, at Departamento Interfacultativo de Bioquimica, Facultad de Medicina, Espinardo, Murcia, Spain. 0003-9861/84 $3.00 Copyright All rights
0 1984 by Academic Press, Inc. of reproduction in any form reserved.
438
oxidative enzymes, and by metalloproteins, has been demonstrated. However, there is disagreement whether the expected products of oxidation are present or involved in physiological processes, as well as whether the reaction is part of a normal or an abnormal metabolic pathway (2). In general, a third minor route has been considered with increasing interest during recent years due to the detection of the antitumoral effect of catecholamines (3), and of dopamine in particular (4). This antitumoral effect for malignant melanoma, in vitro as well as in vivo, has been ascribed to the reactivity of quinones pro-
KINETIC
STUDY
OF DOPAMINE
duced in the oxidation of catecholamines with tyrosinase (3-6). This enzyme was detected in large amounts in malignant melanoma cells (7-9). Oxidative processes of catecholamines by peroxidase (10, 11) or ceruloplasmine (12) via free radical production have been widely studied. However, oxidation by tyrosinase has not been analyzed. Tyrosinase oxidizes diphenols without the formation of free radicals (13,14). The direct product of this enzyme is, therefore, o-quinone, thought to undergo a series of chemical reactions following Raper-Mason’s classic scheme of melanization (15, 16). Tyrosinases from various sources have several properties in common, but also exhibit striking differences; for example, catechol is an excellent substrate for mushroom tyrosinase, but catechol derivatives with substituents in position 4, such as dopamine, noradrenaline, and adrenaline, have much lower oxidation rates. On the other hand, in the case of mammalian tyrosinase, unsubstituted catechol is a poor substrate, and catechol derivatives with the same substituents undergo oxidation at much greater rates (17). The present paper deals with the dopamine oxidation by sodium periodate and mushroom tyrosinase. Spectral characteristics of the oxidation products and their stoichiometry up to dopaminochrome have been established. The kinetic analysis of this pathway has also been studied as an enzymatic-chemical-chemical (E,CC) mechanism, such as described in a previous paper (18). Rate constants for each of the implied chemical steps have been evaluated, and the existence of o-dopaminequinone-H+ as the first intermediate in the enzymatic action is suggested. MATERIALS
AND
METHODS
Mushroom tyrosinase (monophenol monooxygenase, EC 1.14.18.1, 24 DOPA units/mg) and dopamine chlorhydrate were purchased from Sigma. Sodium metaperiodate was analytical grade from Merck. Spectra were recorded with an Aminco DW-2 spectrophotometer with scanning speeds up to 20 rim/s. Reference cuvettes contained, in all cases, all the components except substrate.
OXIDATION
BY TYROSINASE
439
Spectrophotometric measurements were carried out with an Aminco DW-2 spectrophotometer equipped with a Hewlett-Packard recorder with kinetic response, thus allowing the dead recording time to be minimized. When the rate constant of the chemical reaction was greater than 0.1 s-i, an Aminco-Morrow stopped flow accessory equipped with a Dasar TM unit was also used. Dopaminochrome accumulation was spectrophotometrically followed at 480 nm; at this wavelength neither dopamine nor any other possible intermediate present in the reaction medium shows absorbance. The reaction medium was 10 mM phosphate buffer (pH as indicated in each case), 2 mM dopamine (i.e., in saturated conditions), and the amount of enzyme necessary to reach b (& is the time needed to attain the 0.99 Vo/2 rate). In these conditions, absorbance increase was less than 0.2. Determination of intermediate dopaminoquinone-H+ with an absorption maximum at 390 nm was made under the same conditions as described above for dopaminochrome determination. Oxygen consumption was followed by a Rank oxygen electrode (Rank Brothers). Temperature was controlled using a Gilson bath and a Cole-Palmer digital thermistor with a SR f O.l”C. Protein concentration was determined by the Lowry method (19). RESULTS
In an attempt to identify the intermediate compounds generated by dopamine oxidation into dopaminochrome, spectrophotometric determinations were carried out upon oxidation of dopamine by tyrosinase and sodium periodate, respectively. Both compounds oxidize o-diphenols to the respective o-quinones.
Dopamine Oxidation by Tyrosinase Figure IA shows the visible spectra of the products formed in the dopamine oxidation by tyrosinase (12.5 pg/ml) at pH 6.1. Two absorbance maxima (390 and 480 nm, respectively) were detected at pH 6.1, simultaneously developing as the reaction progressed. The graphical analysis of the recordings of Fig. 1A by the matrix method of Coleman et al. (20) corresponded for three absorbing species in solution, as illustrated in Fig. 2A (in Miniprint). When oxidation was carried out at pH 7.0, only the maximum at 480 nm was observed. This suggested that a reaction product
440
JIMENEZ
ET AL.
0.6 A 03
350
450
Akim)
550
L 350
I 150
A(nml
I 550
FIG. 1. (A) Spectrophotometric recordings (from 350 to 550 nm) for the oxidation of dopamine by tyrosinase at 10°C in 10 mM sodium phosphate buffer, pH 6.1. Dopamine (2 UIM) was oxidized with tyrosinase (12.5 pg/mI). Scan speed was 20 rim/s. The first recording was started at 20 s from the beginning of the reaction. (B) Spectrophotometric recordings for the oxidation of dopamine by tyrosinase, with oxygen depletion, from 350 to 550 nm, at 10°C in 10 mM sodium phosphate buffer, pH 6.1. Dopamine (2 mM) was oxidized with tyrosinase (50 fig/ml). Scanning conditions were the same as expressed in (A). (C) Oxygen consumption corresponding to the reaction shown in (B).
was detectable, not at pH 7, but at pH 6.1. This product developed before the maximum at 480 occurred, and agreed with the X,,, established for o-quinone (21). On the other hand, the maximum at 480 nm corresponded to aminochrome (22). It can therefore be assumed that o-quinone is the direct product of the enzymatic action. When dopamine oxidation was achieved by mushroom tyrosinase at pH 6.1, with a large enzyme concentration, it was possible to obtain oxygen depletion in the assay medium within a few minutes of the start of the reaction (Fig. 1C). Figure 1B shows the visible spectrum for this oxidation, undertaken with four times the enzyme concentration used in Fig. IA. As can be seen, the initial recordings were similar to those obtained in Fig. 1A but, from recording No 4, an isosbestic point was defined at 418 nm; this suggested that the enzymatic reaction had stopped due to oxygen depletion. From tracing 4 onward, the transformation of accumulated dopaminequinone-H+ into its products could thus be seen. The graphical analysis for these last recordings (Fig. 2B, in Miniprint) shows that there are two kinetically related species.
Dopamine Oxidation by Periodate It is widely established that o-diphenol chemical oxidation by sodium periodate leads to o-quinone production (22). Figure 3A shows the visible spectrum for the dopamine chemical oxidation, at pH 6.1, with an excess of periodate ([NaIO$[dopamine] = 12.5). The maximum at 390 nm appeared immediately at the beginning of the reaction and then decreased; the maximum at 480 nm subsequently evolved. The appearance of an isosbectic point in these spectra (X = 403 nm) seemed to suggest that there was a transformation of dopaminequinone into dopaminochrome at a constant ratio. This can be confirmed by the matrix method of Coleman et al. (20) (Fig 4A in Miniprint). Figure 3B shows the visible spectrum for dopamine oxidation with insufficient sodium periodate ([NaIOJ[dopamine] = 0.25) at pH 6.1 and, once again, an initial maximum at 390 nm evolved toward another maximum at 480 nm. A new isosbestic point appeared at X = 423 nm. Graphical analysis confirmed that o-dopaminequinone was converted into dopaminochrome at a constant ratio (Fig. 4B, in Miniprint).
KINETIC
STUDY
L50
X(nm)
OF DOPAMINE
OXIDATION
I 350
550
350
“O
BY TYROSINASE
I L50
441
Xtnm)
I 550
hlnml
FIG. 3. Spectrophotometric recordings (from 350 to 550 nm) for the oxidation of dopamine by sodium periodate at 10°C in 10 mM sodium phosphate buffer, pH 6.1. Scan speed was up to 20 rim/s. the first scan was started at 40 s from the beginning of the reaction. (A) Dopamine (0.4 mM) was oxidized with 2.5 mM NaIO,. (B) Dopamine (2 mM) was oxidized with 0.5 mM NaIOl. (C) Dopamine (0.2 mM) was oxidized with 0.4 mM NaIO,.
When oxidation was carried out, with an equimolar ratio, until an absorbance increase could no longer be detected (Fig. 5), the existence of two compounds, aminochrome with two absorbance maxima (300 and 480 nm), and dopamine, with a maximum at 280 nm, was evident. This suggested that dopamine was present in the reaction medium. The addition of a new equivalent of periodate to the reaction mixture increased the value of absorbance at both 300 and 480 nm twofold. Two equivalents of periodate were therefore necessary to render an equivalent of dopaminochrome. These results, together with those shown in preceding figures, suggested a
set of steps as shown in Fig. 6, agreeing with the recently postulated pathway for L-DOPA oxidation by tyrosinase (23). Stoichiometry of Dopamine by Tyrosinase
Oxidation
To establish the stoichiometry of the dopamine oxidation processes, an assay was performed by following the absorbance increase at pH 6.5 and 15°C (Fig. 7) with the reaction time at 390 and 480 nm, respectively. The rate of enzymatic reaction was evaluated as the tangent, at t = 0, to the recording of absorbance at 390 nm, (curve B), and also as the slope of the linear zone of the dopaminochrome
JIMENEZ
442
ET AL.
0.17
A
0.085
Xhm) FIG. 5. Oxidation of dopamine by sodium periodate at 15’C in 10 mM sodium phosphate buffer, pH 6.5. Dopamine (0.4 mM) was reacted with 0.4 mM (A) and 0.8 mM (B) NaIO,.
accumulation curve (curve A). The o-quinone production rate was twofold greater than the dopaminochrome accumulation rate, when dopaminochrome was accumulated at constant rate. These results agree with the postulated mechanism for the steps that follow the enzymatic reaction (Fig. 6). Kinetic
Approach
According to the postulated pathway previously described, dopamine oxidation into dopaminochrome by tyrosinase can
T~rosinase
H
0
I
FIG. 6. Pathway proposed dopamine to dopaminochrome nase.
for the oxidation of catalyzed by tyrosi-
FIG. 7. Product formation (with time) for 2 mM dopamine oxidation with tyrosinase (10 pg/ml) at 15”C, pH 6.5. (A) Absorbance increase of dopaminochrome accumulation followed at 480 nm. (B) Absorbance increase of dopaminoquinone followed at 390 nm.
be considered as a mechanism of two chemical reactions coupled to the enzymatic step (mechanism E,CC); note the recycling, by a set of chemical reactions, of a product of the enzymatic reaction into a substrate of this same step, according to the expressed scheme in Fig. 7 and developed theoretically in the Appendix. Evaluation of the Apparent Rate Constant Dopamine oxidation to dopaminochrome by tyrosinase presented a marked lag period, as has previously been shown (Fig. ‘7). This lag period was independent of enzyme concentration, in accordance with the theoretical approach developed for the scheme of chemical reactions coupled to an enzymatic step (Mechanism E,CC), with the particular characteristics of the proposed pathway for this oxidative process. On the other hand, dopaminochrome accumulation must reach a steady-state condition, where the accumulation is half the enzymatic velocity for the dopamine conversion into dopaminoquinone (Eq. [lo] of Appendix). Dopaminochrome accumulation recordings at different enzyme concentrations are presented in Fig. 8. The resulting curves tend toward straight
KINETIC
0
60
STUDY
t Is)
OF DOPAMINE
OXIDATION
BY TYROSINASE
443
120
FIG. 8. (A) Dopaminochrome accumulation with time at pH 6.2 and 15°C followed by absorbance increase at 480 nm for different enzyme concentrations (rg/ml): (a) 2.5, (b) 4, (c) 8, and (d) 10. (B) Plot of dopaminochrome accumulation rate, under steady-state conditions, against enzyme concentration.
lines, with slopes directly related to the enzyme concentration (Fig. 8B), and their lag periods (defined as the intercept on the abscissa axis of the tangent, at t co, to the dopaminochrome accumulation curve), were independent of enzyme concentration (Fig. 8A), and can be assimilated to l/k,, (eq. [ll] of Appendix); therefore, kapp = 0.042 s-‘. This constant can also be obtained from the analysis of the accumulation curves with the reaction time (Eq. [12] of Appendix). Figure 9 shows an analysis for the curves of Fig. 8 according to that equation. From the slope of these lines an apparent constant with a significantly similar value (0.04 s-l) to the calculated value from the lag period has been evaluated; thus, kap values will always be calculated from the lag period measurements.
Variation of the Lag Period with pH and Temperature: Evaluation of the Specific Rate Constants The lag period presented a significant variation with pH and temperature. As can be observed in Table I, the lag period decreased for pH and temperature values between 6 and 7, and 10 and 35”C, respec-
FIG. 9. Semilogarithmic representation of (PI - [D&J against time, according to Eq. [12]. The values of [D] correspond to those of Fig. 1A.
tively, when the pH and temperature were increased. If the kinetic treatment, obtained by approximation to equilibrium, was acceptable for this system, a linear relation must exist between the lag period (twofold) and proton concentration, at a fixed temperature. From the linear regression-fitted plots, kc can be evaluated from the slope, and kl from the intercept on the ordinate axis (Fig. 10). The firstorder rate constants for deprotonation of dopaminoquinone-H+ to dopaminoquinone and for the cyclization of dopaminoquinone to leukodopaminochrome at different temperatures were expressed in Table II.
TABLE
I
RECIPROCALS OF THE FIRST-ORDER RATE CONSTANTS FOR THE CONVERSION OF DOPAMINOQUINONE-H+ INTO DOPAMINOCHROME AT SEVERAL PH AND TEMPERATURE VALUES l/k, pH/t (“C) 6.1 6.4 6.7 7.0
10
15
44.5 31.5 16.7 11.5
31.5 17.8 12.5 7.2
20 16
10 6.5 3.7
(s) 25 10 5.7 3.7 2.2
30 6 3.8 2.2 -
35 3.6 2.1 1.25 -
444
JIMENEZ
ET AL. 1
7
100 2 k&is)
K,b) 5-1 50
\1 0.1
0.4 [H'] @I
3.6
"*
FIG. 10. Plot of the reciprocals of observed rate constants against proton concentrations at different temperatures, in the process of conversion of dopaminequinone-H+ to dopaminochrome (see Eq. [ll]).
FIG. 11. Arrhenius plot for k, and kl, values of k, and kl correspond to those of Table II.
The value of the second ionization constant for dopamine [pK, = 10.6 (24)] should be preliminarily considered as the ionization constant for dopaminequinone-H+. The results obtained correspond closely to the theoretical solutions used to resolve the set of chemical reactions coupled to the enzymatic oxidation of dopamine by tyrosinase.
zation processes, Arrhenius plots were made using the k,- and kl-obtained values, as shown in Fig. 11. The calculated activation parameters for the cyclization process, as well as for deprotonation, are summarized in Table III; the high value for AS of the cyclization process can be attributed to a very favorable probability factor that should lead to a very fast cyclization.
Evaluaticm of Themnodynamic Parameters for the Deprotonation and Cgclization Processes For the evaluation of the activation energies for the deprotonation and cycli-
DISCUSSION
o-Dopaminequinone has been detected as the first product of tyrosinase action on dopamine, at pH conditions where the quinone was stabilized by the protonation of its amine group. This group acts as a nucleophillic group during the intramoTABLE II lecular reaction of cyclization to leukoaminochrome (addition Michael 1, 4). DoFIRST-ORDER RATE CONSTANTS (kI) FOR DEPROTONAis more stable than doTION OF DOPAMINOQUINONE-H+ TO DOPAMINOQUINONE paminequinone pH because it AND FOR THE CYCLIZATION OF DOPAMINOQUINONE TO paquinone at physiological has a greater value of pK, for its amine LEUKODOPAMINOCHROME AT VARIOUS TEMPERATURES group [dopamine, 10.2; dopa, 8.72 (24)]. kl This means that there is a smaller ratio (sf). r”c, (0 for deprotonated dopamine. The internal cyclization reaction is therefore less fa10 0.06 417 vored. These results agree with the greater 15 0.11 588 cytotoxicity for the dopamine tyrosinase 20 0.19 1162 system (5). 25 0.39 1811 On the other hand, o-quinone stability 30 0.46 3187 depends on pH. At pH 6.1, the quinonic 35 0.84 5312 intermediate was accumulated to a level “Derived from Eq. [ll] by using a value of KS detectable by graphical analysis (Fig. 1; = 2.51. lo-“. and Fig. 2 in Miniprint). The system
KINETIC
STUDY
OF DOPAMINE
OXIDATION
TABLE
E Deprotonation Cyclization
mol-‘)
445
III
THERMODYNAMIC ACTIVATION PARMETERS FOR THE DEPROTONATION DOPAMINOQUINONE AND FOR THE CYCLIZATION OF DOPAMINOQUINONE
(kcal
BY TYROSINASE
OF DOPAMINOQUINONE-Hi INTO INTO LEUKODOPAMINOCHROME
MS (kcal
mol-‘)
A’3 (kcal
mol-‘)
As* WJ)
18.3
17.7
18
-1
18.5
17.9
13
16.4
reaches the steady-state and dopaminochrome is accumulated, after a lag, at constant rate (curve A of Fig. 7). From the experiments of dopamine oxidation by sodium periodate (Fig. 5), two equivalents of periodate were considered necessary for the complete oxidation of one equivalent of dopamine to dopaminochrome. In the experiments undertaken with tyrosinase (Fig. ‘7), the rate of dopaminochrome accumulation was half that of o-dopaminoquinone-Hf. Both experiments allowed us to conclude that the stoichiometric equation for the conversion of o-quinone-H+ into aminochrome is 2 odopaminoquinone-H+ - dopamine + dopaminochrome. When dopamine oxidation was carried out with insufficient sodium periodate (Fig. 3B), an isosbestic point at 423 nm could be seen. Analysis of the spectra showed that there were two species (Fig. 4B of Miniprint). However, there are four kinetically related species: dopamine, o-dopaminequinone-H+, leukodopaminochrome, and dopaminochrome. This result can be explained by considering that the leukodopaminochrome concentration was too small to be detected by matrix analysis. This coincides with the great difference of oxidation-reduction potentials between the o-quinone/o-diphenol and halochrome/leukohalochrome pairs (25). Furthermore it should be taken into account that, during the whole reaction, 1 mol dopamine and 1 mol dopaminochrome originate at a constant ratio from 2 mol dopaminequinone-H+. A similar situation occurs in dopamine oxidation with very high concentrations of tyrosinase. In these conditions, the oxygen was depleted in the medium; from tracing No.
4 in Fig. lB, the test for two species was positive, and was assumed to be o-dopaminequinone-H+ and dopamine plus dopaminochrome (Fig. 2B of Miniprint). The experiments performed with sodium periodate in excess (Fig. 3A) showed an isosbestic point, different from the preceding one, at 403 nm, which can be explained by the transformation of dopaminequinone-H’ into dopaminochrome with a 1:l stoichiometry, since the excess sodium periodate prevents the accumulation of dopamine in the medium. Graphical analysis for two species was therefore applicable, odopaminequinone-H+ and dopaminochrome being identified. When dopamine oxidation by periodate was carried out under stoichiometric conditions, the cyclization and oxidation reactions took place simultaneously, so no isosbestic point occurred (Fig. 3C) and graphical analysis determined three species which were considered to be dopamine, o-dopaminequinone-H+, and dopaminochrome. An identical result was obtained with low amounts of tyrosinase, when the oxygen in the solution was not depleted (Fig. 1A; and Fig. 2A of Miniprint). A pathway, the postulated steps of which are different from those proposed by Lsvstad (26), can therefore be proposed (Fig. 7), since Lsvstad assumed the appearance of free radicals in dopamine oxidation by ceruloplasmine. Kinetic studies also confirmed that dopamine oxidation to dopaminochrome followed the same pathway previously proposed by us for LDOPA (18), and also established that odopaminequinone-H+ was more stable than o-dopaquinone-H+ at physiologic PH (5).
446
JIMENEZ APPENDIX
The postulated be schematically
ET AL.
and
mechanism in Fig. 6 can written as follows:
[31 By using the fractional concentration tor of Cha (28), fB, as,
kl
PI fB = [BH] + [B] = where A = dopamine, BH = dopaminequinone-H+, B = dopaminequinone, C = leukodopaminochrome, and D = dopaminochrome. The equation for dopaminochrome accumulation curve can be obtained if it is assumed that (i) the enzymatic reaction rate remains constant (V,), since the variation of dopamine concentration during the reaction time is very small. (ii) The constant (K) for the equilibrium BH + C P A + D is very high, in accordance with the electrochemical data (5). It can thus be seen that the leukodopaminochrome concentration in the reaction medium tends toward zero throughout the whole reaction. Therefore, the apparent rate constant of leukodopaminochrome decomposition is the same as that of formation (kJB]) at all times. (iii) The rate constants for the steps BH P B - C are high, as they correspond to the deprotonation and cyclization processes. It can thus be assumed that they are in pseudo steady state. A simplified model can therefore be formally written as shown in the following scheme, in agreement with the kinetical schemes resolution, where one part is in steady state and the other in presteady state conditions (27).
kpJH+] + kl + k, ’ ‘41
the differential equations model can be written as 2
2kc fB.
In view of the preceding considerations, we can write: = kl[BH] - (kJH+]
+ kJ[B] = 0, [l]
where [X] = [BH] + [B] + [C] li [BH] + [B]
PI
and
Integrating these equations, and taking into account Eq. [4] and that [X] = 0 at t = 0, the following equation
v, “I
= (2k,kl)/(k-,[H+]
+ kl + kc) %k.
X
1 -
e
k-dH+l+k,+S
PI
t >
can be established, where the apparent rate constant is defined as
2We Kap = k-,[H+] + kl + kc ’ rD1 = :
it + (2k,kc)/(k-,[A+] X e- k-iH+l+h+k
PI
+ kl + kc)
t 1
+ kl + kc)>
PI
for t - co, it is possible to define how D is accumulated when the system reaches its steady state,
A&X
F
of simplified
= v, - ZfBkdx]
- (2klk,)/(k_JH+] -A
fac-
[2]
ID”
= :
tt - (2k,%)/(ke,[;+]
+ kl + kc>*
[W The lag period (L) can be defined as the intercept on the abscissa of the straight zone prolongation of the D accumulation curve,
KINETIC
STUDY
OF DOPAMINE
2L~[H’l+L+L2 K&c k1 kc ka, ’
WI
where K,=$. 1
A plot of 2L against [H+] gives a straight line, the slope of which gives the value of kc, if K, is known, and the intercept on the ordinate axis the kl values. On the other hand, using Eqs. [g] and [9], it can be established that
WDI - PISS)= In
-
V,WdH+l+ h + kc) 2kch 2Ul t. [12]
k-,[H+] + kl + kc Hence a plot time gives a which enables rate constant
of ln([D] - [D],,) against straight line,-the slope of us to evaluate the apparent (kap).
ACKNOWLEDGMENTS This research has been partially supported by a grant from the Comision Asesora de Investigation Cientifica y Tecnica. M. Jimenez had a fellowship from Ministerio de Education y Ciencia. REFERENCES 1. MOLINOFF, P. B., AND AXELROD, J. (1971) Annu. Rev. Biochem 40, 465-500. 2. HARRISON, W. H. (1963) Arch. Biochem. Biophys. 101, 116-130. 3. WICK, M. M. (1980) Cancer Res. 40, 1414-1418. 4. WICK, M. M. (1979) J. Natl Cancer Inst. 63,14651467. 5. WICK, M. M. (1979) Cancer Treat. Rep. 63, 991997. 6. WICK, M. M. (1978) J Invest. DermatoL 71. 163164.
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BY TYROSINASE
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7. POMERANTZ, S. H., AND LI, J. P.-C. (1973) Yale J. Biol Med 46, 541-552. 8. NISHIOKA, K. (1978) Eur. .I Biochem 85, 137146. 9. BURNETT, J. B. (1971) J. Bid Chem 246, 30793091. 10. VAN WOERT, M. H., AND AMBANI, L. M. (1974) A&v. Neural. 5, 215-223. 11. I&STAD, R. A. (1979) Gen Pkarmaco~ 10, 437440. 12. WALLAS, E., AND WALLAS, 0. (1961) Arch Biochem Biophys. 95,151-162. 13. SEIJI, M., SASAKI, M., AND TOMITA, Y. (1978) Tohoku K. Exp. Med. 125,233-245. 14. TOMITA, Y., SEIJI, M., AND IGARASHI, A. (1980) J. Invest. Dermatol 7. 377-380. 15. RAPER, H. S. (1928) Physiol. Rev. 8. 245-282. 16. MASON, H. S. (1948) J. Biol. Chem. 172, 83-99. 17. YASUNOBU, K. T. (1953) in Pigment Cell Biology (Gordon, M., ed.), p. 583, Academic Press, New York. 18. GARCIA-CARMONA, F., GARCIA-CANOVAS, F., IBORRA, J. L., AND LOZANO, J. A. (1982) B&him Bie phys. Acta 717, 124-131. 19. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. BioL Chem. 193. 265-275. 20. COLEMAN, J. S., VARGA, L. P., AND MASTIN, S. H. (1970) Iwg. Chem 9. 1015-1020. 21. YASUNOBU, K. T., PETERSON, E. W., AND MASON, S. H. (1959) J. Biol. Chem 234, 3291-3295. 22. GRAHAM, D. G., AND JEFFS, P. W. (1977) J. BioL Chem. 252, 5729-5734. 23. GARCIA-CANOVAS, F., GARCIA-CAF~MONA,F., IBORFLA, J. L., AND LOZANO, J. A. (1982) J. Biol Chem. 257, 8738-8744. 24. GRAY, D. O., AND WEITZMAN, P. D. J. (1968) in Data for Biochemical Research (Dawson, R. M., Elliot, D. C., Elliot, W. H., and Jones, K. M., eds.), pp. 22-23, Oxford Univ. Press (Clarendon), London/New York. 25. SWAN, G. A. (1974) Forts&. Chem Org. Naturst. 31, 521-582. 26. L&STAD, R. A. (1971) Acta Chem. Stand 25, 3144-3150. 27. RICARD, J., But, J., AND MEUNIER, J. C. (1977) Eur. J. Biochem. 80,581-592. 28. CHA, S. (1968) J. BioL Chem 243, 820-825.
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