The Role of pH in the Melanin Biosynthesis Pathway*

Having oxidized 3,4-dihydroxyphenylalanine (dopa) with sodium periodate or mushroom tyrosinase in a pH range from 3.5 to 6.0, it has been possible to detect spectrophotometrically 4-(2-carboxy-2-aminoethyl)- 1,2-benzoquinone with the amino group protonated (o-dopaquinone-H’), a postulated intermediate in the melanogenesis pathway. When the pH value was greater than 4, the final product obtained was 2-carboxy-2,3-dihydroindole-5,6-quinone (dopachrome); however, for pH values lower than 4, two different products were identified by means of cyclic voltammetry: 5-(2-car-boxy-2-aminoethyl)-2-hydroxy-1,4-benzoquinone and dopachrome. These products appeared when oxidation was achieved with the enzyme as well as with perio- date. This suggests that two chemical pathways can arise from o-dopaquinone-H’, whose relative impor- tance is determined by the pH. The steps of these pathways would be dopa -+ o-dopaquinone-H+ -+ o-dopa- quinone -+ leukodopachrome -+ dopachrome, for the first one, and dopa -+ o-dopaquinone-H+ + 2,4,5-trihy- droxyphenylalanine * 5-(2-carboxy-2-aminoethyl)-2-hydroxy-1,4-benzoquinone


very slowly
dopa oxidation by tyrosinse (5, 6). The existence of dopaquinone-H+ as an intermediate was inferred from the action of tyrosinase on a variety of catechols. Evidence has also been presented for the formation of a dopaquinone analogue upon oxidation by tyrosinase of tyrosine-containing oligopeptides in which the a-amino group of tyrosine is substituted (7). The other intermediate in the pathway, named leukodopachrome, has only been observed as a product of dopachrome reduction (5).
The identification of topa as a product of the action of tyrosinase on tyrosine (8) has led to some authors' attempts to include this product in the melanin biosynthesis pathway.
Taking into account these results as well as the graphical analysis data, Graham and Jeffs ( 5 ) proposed another possible pathway that included topa as an intermediate: tyrosine -+ dopa + topa "-f 5-(2-carboxy-2-aminoethyl)-4-hydroxy-1,2benzoquinone -+ p-topaquinone + intermediate compound "+ dopachrome. Neither of these authors consider the appearance of o-dopaquinone-H', but they stated that tyrosine should be directly converted into topa as a result of tyrosinase action and that topa would be converted into its quinones.
On the other hand, a consideration of oxidation-reduction potentials for the pairs o-dopaquinone-H'/dopa and dopachrome/leukodopachrome led to the proposal by Lerner and Fitzpatrick (1) that leukodopachrome could be oxidized into dopachrome by 0-dopaquinone-H'. Nevertheless, this suggestion has not been taken into account (5,6,11). The great instability of the intermediates in this pathway as well as the difficulty of their isolation also hindered their study.
In the present study, dopa was oxidized by sodium periodat,e and tyrosinase for several different pH values (3.5-6.0) in an attempt t.o determine whether o-dopaquinone-H' and topa could be considered as intermediates in the pathway of melanin synthesis. It is proved that the pathway is dependent on pH with regard to its intermediate products. When reactions are achieved at neutral or slightly acid pH, the Raper-Mason pathway ( Fig. 2A) is the main one. However, for strongly acid pH values lower than 4, two pathways should compete from o-dopaquinone-H' (Fig, 2B). The first one is similar to that described in Fig. 2A and should include the deprotonation of 0-dopaquinone-H', yielding o-dopaquinone; the cyclization of the latter should give leukodopachrome, whose oxidation by o-dopaquinone-H' should give dopachrome and dopa. The alternative pathway, that includes p-topaquinone formation, should be the most important for lower pH values; this pathway would suppose the addition of water to the quinonic ring to yield topa, which is oxidized by o-dopaquinone-H', with formation of dopa and p-topaquinone, with a very slow evolution of this last compound to dopachrome.
Two considerations are proposed that must be borne in mind with regard to the Raper-  (12).

RESULTS
In an attempt to define whether or not o-dopaquinone-H' and topa are intermediates in the melanin biosynthesis pathway, their spectrophotometric detection was carried out upon oxidation of dopa by sodium periodate and tyrosinase. a t 390 nm that was ascribed to o-dopaquinone-H+, in agreement with Graham and Jeffs (5); this peak shifted with time towards another one of X , , , at 475 nm, characteristic of dopachrome. Recordings are present in Fig. 3A showing an isosbestic point at X = 416 nm whose appearance suggested, as a first approximation, the occurence of two species kinetically related.
The graphical analysis of the recordings in the visible spectrum by the matrix method of Coleman et al. (13) gave a good fit for two absorbing species in solution, as is shown in Fig. Nevertheless, as will be discussed later, in this case three kinetically related species should exist.
The appearance of the isosbestic point seems to suggest that an equivalent quantity of dopa to that of periodate has been converted into o-dopaquinone-H', which in turn yields dopachrome. In this way, Fig. 5 shows a plot of maximal concentrations of o-dopaquinone-H+ at the beginning of the reaction against periodate concentrations; a straight line of slope 1 was obtained (c$~''''onr~Ht = 1250 M" Cm" (14) suggesting that periodate was fully consumed following a 1:l stoichiometry. In this case, it seemed interesting to study whether oxygen does participate in the conversion of leukodopachrome into dopachrome, following the scheme of Raper (2) (Fig. 1). However, oxygen consumption through reaction time was measured in the same experiments, and no consumption was detected.
Bearing in mind the considerations of oxidation-reduction potentials for the pairs o-dopaquinone-H+/dopa and dopachrome/leukodopachrome indicated by Lerner and Fitzpatrick (1) and assuming that in the experimental conditions described above leukodopachrome was not oxidized by sodium periodate, the latter being fully depleted previously, nor by oxygen as it does not participate in this step, investigation was made to ascertain whether leukodopachrome oxidation was achieved by o-dopaquinone-H+. Thus, concentration of dopachrome was measured when it reached a constant value; it was found that this concentration was half of the periodate concentration and therefore half that of o-dopaquinone-H'. These results suggested that o-dopaquinone-H' actually oxidizes leukodopachrome, being reduced to dopa. The slope of the straight line resulting from the plot of dopachrome concentration versus periodate concentration was also equal to 0.5 (Fig. 5).
When concentration of sodium periodate was greater than

a set of recordings was
obtained which showed the presence of two isosbestic points, at X = 362 nm and X = 398 nm, as is shown in Fig. 3B. The graphical analysis of the spectra gave a good fit for two absorbing species in solution, kinetically related (Fig. 4B). Finally, when the oxidation was carried out in equimolar conditions, [dopa]/[NaIO,] = 1, no isosbestic point appeared, as can be seen in the recordings of Fig. 3C. The graphical analysis of the spectra showed the presence of three absorbing species kinetically related (Fig. 4C).

Melanin Biosynthesis
Oxidation by Tyrosinase-When the oxidation of dopa was another peak at 475 nm corresponding to dopachrome. In this achieved by mushroom tyrosinase for slightly acid pH values, case, no isosbestic point was defined, as can be observed in it was again possible to record a peak at 390 nm that was Fig. 6A; the analysis of the different recordings gave a good fit coincident with the maximum obtained previously upon oxifor three absorbing species kinetically related (Fig. 7A). dation by periodate. This maximum has been related to 0-When the same assays were performed, but the absorbance dopaquinone-H' and is shifted during reaction time to give increase with time was recorded at a fixed wavelength, the m e l a n i n s c + c  following results were obtained. When the recording was made at X = 390 nm, corresponding to the maximum of o-dopaquinone-H+, an apparent burst was detected (Fig. 8B). However, when the recording was made at X = 475 nm, a wavelength coincident with the dopachrome maximum, a lag time appeared (Fig. 8A). This lag period is not dependent on the enzyme concentration, but it is due to the series of chemical reactions arising between o-dopaquinone-H' and dopachrome (Fig. 2 4 ) ; and thus, it is dependent on pH. This lag period has the value of l / k , where Iz is the rate constant of the conversion of o-dopaquinone-H' to d~pachrome.~ Note that o-dopaquinone-H*, the direct product of the enzymatic reaction, was accumulated following an apparent burst and the reaching of a steady state (Fig. 8C).
The kinetic analysis of a system of chemical reactions coupled to an enzymatic reaction4 predicts that the rate of formation of the final product, when the system has reached the steady state, should be the same as the initial velocity of the enzymatic step provided that the chemical reactions are of fist order or pseudo-fist order; when an intermediate undergoes a second order reaction, this rate is half of the initial velocity of the enzyme. It is important to point out that, in the first case, the stoichiometry would be 1:1, while it would be 2:l in the second case. From the results presented in Fig.  8, it is possible to calculate that the rate of dopachrome accumulation in the steady state is half of the initial velocity of o-dopaquinone-H+ formation by the enzyme, suggesting that a 2:l stoichiometry must exist in the pathway of coupled chemical reactions with respect to dopachrome formation.
Similar assays were performed with a 5-fold greater enzyme amount so that oxygen was depleted in the solution. In these conditions, it was possible to determine the same isosbestic point that appeared for a [dopa]/[NaIO,] ratio of 4.4, and the matrix analysis applied to this anoxia situation showed the occurrence of two species kinetically related. However, as well as in the experiment shown in the Fig. 3A, three species are actually present, as will be discussed later. On the other hand, the lack of participation of oxygen in the oxidation of leukodopachrome into dopachrome was verified since the amount of oxygen in solution was determined to be null (starting from trace 6 of Fig. 6B).
Cyclic Voltammetry-The two pathways of oxidation of dopa proposed by Graham and Jeffs (5) that can be catalyzed by tyrosinase lead to p-topaquinone and dopachrome. Since the Xmax for these compounds are very similar (485 nm and 475 nm), it is difficult to determine spectrophotometrically whether the two products could be formed at the same time for the same pH values. However, cyclic voltammetry allows  Fig. 4C was applied. B, tyrosinase concentration, 0.1 mg/ml. The same test as in Fig. 4A was applied, starting in tracing number 6. The meanings of different magnitudes of graphical analysis are defined in Fig. 4 (see miniprint). a possible approach to this problem. When oxidation of dopa was achieved by means of tyrosinase or sodium periodate for pH values greater than 4, cyclic voltammograms (Figs. 9 and 10) showed, respectively, a cathodic peak and an anodic peak. The cathodic peak arose from the dopachrome reduction to leukodopachrome, while the anodic peak appeared for a potential value slightly more positive and should be explained by the oxidation of leukodopachrome into dopachrome (15). However, cyclic voltammograms obtained for pH values lower than 4, both for tyrosinase and sodium periodate, were constituted by two cathodic peaks and two anodic peaks, respectively (Fig. 11, A and B).
The cathodic peak obtained for the more negative potential of -0.10 V uersus SCE arose from the reduction of dopachrome into leukodopachrome; its corresponding anodic peak appeared at 0.05 V versus SCE and was due to the oxidation of leukodopachrome into dopachrome. The cathodic peak located at a more positive potential, 0.05 V versus SCE, can be ascribed to the reduction ofp-topaquinone into topa, which shows an anodic peak at the potential value of 0.12 V versus SCE, corresponding to the oxidation of topa into p-topaquinone, as was assessed by reference to the peaks obtained in the cyclic voltammograms of dopachrome and topa recorded separately for the same pH value. The separation between the potentials of the oxidation and reduction peaks is almost equal to the theoretical value for reversible processes.
Leukodopachrome-Leukodopachrome is another intermediate in the pathway leading to dopachrome. The graphical   N d 0 4 (B). The enzyme was incubated with the substrate and then the solution was located into the cell with the proper electrodes. Nitrogen was bubbled through the solution before starting the record. The magnitude I is the current intensity expressed in microamperes and E is the voltage expressed in volts (V). Working electrode: hanging mercury electrode; scan rate, 0.5 V.s".

DISCUSSION
The identification of intermediates in the pathway of melanin biosynthesis has been hard to establish because of the great reactivity of these short half-life compounds. Two intermediates have been proposed for the steps included between dopa and dopachrome, namely o-dopaquinone-H' and leukodopachrome.
The occurrence of a maximum centered a t 390 nm when tyrosinase acts on tyrosine (linked to other amino acids by means of its a-amino group) led to the proposal that o-dopaquinone-H' was the product of the enzymatic action. Nevertheless, identification of this compound as the direct product of the enzyme action on dopa has never been achieved (5, 6).
It was possible to observe the appearance of o-dopaquinone-H' working with tyrosinase at acid pH values from 3.5 to 6.0 and with a rapid scan spectrophotometer (Fig. 6).
As is shown in the scheme of Fig. 2 A , the formation of dopachrome from o-dopaquinone-H+ is achieved by the cyclization of the molecule following a Michael intramolecular 1,4 addition; this is only possible when the amino group is unprotonated so that the reaction is dependent on pH. For pH values close to 7, the o-dopaquinone-H' intermediate could not be detected by means of graphical analysis due to the rapidity of cyclization and the consequently very low concentration of the intermediate. For lower pH values (less than 6), the reaction became greater, making possible its detection by graphical analysis (Fig. 7A). As time elapsed, the system reached a steady state, and dopachrome was accumulated after a lag period a t a constant rate (Fig. &A).
From the experiments of dopa oxidation by sodium periodate with dopa in excess (Fig. 5), where no oxygen consumption was detected and where it was determined that the amount of dopachrome formed was half that of o-dopaquinone-H', and from the experiments realized with tyrosinase (Fig. 8), where it can be seen that the rate of dopachrome accumulation is half that of o-dopaquinone-H', it can be inferred that the following reaction should occur:  In view of the preceding consideration, we believe that the potential differences between the pairs o-dopaquinone-H'/ dopa and dopachrome/leukodopachrome pointed out by Lerner and Fitzpatrick (1) should be borne in mind and that this step should be added as a n obligatory one to the pathway of melanization.
When the oxidation of dopa is achieved in defect of sodium periodate, the appearance of an isosbestic point a t X = 416 nm can be seen, being &Fhrome = 1.800 M" cm" (Fig. 3A). When the analysis of the spectra were performed, two species were detected (Fig. 44). However, from the considerations outlined above, it can be inferred that three species kinetically related are actually present: dopa, o-dopaquinone-H', and dopachrome. In fact, from 2 mol of o-dopaquinone-H', 1 mol of dopa, and another of dopachrome were released in a constant ratio so that the analysis detected these compounds as a single species. A similar situation can be obtained with the enzyme tyrosinase when the reaction is achieved with very high concentrations of tyrosinase. In this way, the oxygen was depleted in the medium, and the graphical analysis at short times gave a good fit for three species: dopa, o-dopaquinone-H', and dopachrome. Nevertheless, from the number 6 tracing in Fig.   6B, the test for two species was accomplished: o-dopaquinone-H' and dopachrome (Fig. 7B).
The experiments performed with sodium periodate in excess showed the appearance of an isosbestic point different from the preceding one, at X = 398 nm, ~$ r~~ = 1.100 M" cm" (Fig. 3B), that can be explained by the transformation of odopaquinone-H+ into dopachrome following 1:1 stoichiometry. The excess of periodate prevents the accumulation of dopa in the medium, and this excess can also oxidize the leukodopachrome formed, preventing the action of o-dopaquinone-H' as an oxidant. In this case, the graphical analysis (Fig. 4B) gave a good fit for two species: o-dopaquinone-H+ and dopachrome.
When the oxidation is performed in stoichiometric conditions for dopa and periodate, the cyclization and oxidation reactions take place at the same time; periodate can also oxidize to leukodopachrome so that no well defined isosbestic point appears (Fig. 3C). The graphical analysis was in accord-ance with three species (Fig. 4C) which, in agreement with Graham and Jeffs (5), are supposed to be dopa, o-dopaquinone-H+, and dopachrome. The same result is obtained with low amounts of tyrosinase provided that the oxygen in the solution is not depleted (Fig. 6 and 7 , A ) .
The third intermediate of the pathway leading to dopachrome is leukodopachrome. This compound can be a substrate for the enzyme. However, the possibility that the enzyme may act again in this step is negligible since high concentrations would be required in order to compete with dopa. In these conditions, leukodopachrome would be detected by graphical analysis, and this was never the case in the present study.
Assays performed for pH values lower than 4 allow investigation as to whether topa participates in the melanin biosynthesis pathway, following a different approach than that proposed by Graham and Jeffs (5). These authors proposed a minority melanogenesis pathway that includes the participation of topa, suggesting that this compound should arise from the direct action of the enzyme on dopa and should be transformed later into p-topaquinone. So, the participation of o-dopaquinone-H' is not considered in this scheme.
We have always detected the formation of o-dopaquinone-H' as the first oxidation product, irrespective of pH value. Nevertheless, the formation of p-topaquinone from o-dopaquinone-H+ can be achieved by means of solely chemical reaction. For strongly acid pH values, the cyclization reaction of o-dopaquinone-H+ is very slow due to the distance of the pK range for the amino group. In these conditions, the addition of water to the ring can take place with the formation of topa, which in turn can be oxidized by o-dopaquinone-H+, leading to p-topaquinone that slowly yields dopachrome (5) (Fig. 2B). This pathway would compete with the cyclization one, and the relative importance of each one would be dependent on pH.
As we pointed out previously, the maxima for p-topaquinone and dopachrome are very near so that their spectrophotometric identification becomes very difficult. Nevertheless, in the results obtained in cyclic voltammetry experiments, the peak potentials are separate enough as to allow the identification of these compounds. Cyclic voltammograms (Fig. 11A) showed the formation of two products when we worked with tyrosinase with strongly acid pH values (3.5); the correspondent peaks were ascribed top-topaquinone and dopachrome by means of comparison with the standards of these compounds. The evidence that these products are not formed directly by the enzyme arises from the fact that both p-topaquinone and dopachrome were obtained when the oxidation of dopa was performed with sodium periodate (Fig. 1lB).
Cyclic voltammograms also showed that for pH values greater than 4.0 only dopachrome was obtained as final product when the oxidation was achieved by tyrosinase as well as by sodium periodate (Figs. 9 and 10, A and B). It must be noted that topa does not accumulate in the medium because this product is in a similar situation to that of leukodopachrome, and thus, it is rapidly oxidized by o-dopaquinone-H'. Topa has also been obtained by reduction of p-topaquinone with NaBH4 (5).
Our experiments also would explain the initial results obtained by Lissitzky and Rolland (8) with respect to the topa synthesis by tyrosinase action on tyrosine. These authors worked in unbuffered aqueous solution; hence, the addition of ascorbic acid should decrease the pH of the reaction medium, enhancing the addition of water into the ring. On the other hand, cyclization should be prevented by the o-dopaquinone-H+ reduction by ascorbic acid, and in this way, only the topa formation would progress.
From a l l the preceding considerations, it can be inferred as a conclusion that the formation of intermediates in the melanin biosynthesis pathway is dependent on pH. The o-dopaquinone-H+ is always the direct product of the enzymatic action of dopa, and from this compound, two competitive pathways arise, the one yielding dopachrome and the otherptopaquinone. The pathway of p-topaquinone (Fig. 2B) is the major one for very acid pH values, while for a physiologic pH, the pathway of dopachrome formation (Fig. 2 A ) is the main one.