Pigment Cell Research Suppl. 2: 25-31 (1992)
The Role of Peroxidase in Melanogenesis Revisited GIUSEPPE PROTA Department of Organic and Biological Chemistry, University of Naples, Via Mezzocannone 16, 1-80134 Naples, Italy
Historically, our concept of melanogenesis developed from the early observation of Bourquelot and Bertrand (1) that tyrosinase was able to effect the oxidative conversion of tyrosine to a black insoluble pigment closely resembling natural melanins. This and in v i tro studies led subsequent gradually to a view of melanogenesis as a biochemically simple process involving the sole interaction of tyrosinase with the primary substrate tyrosine. However, as knowledge of the chemistry of melanin pigmentation has improved in more recent years, it has become increasingly clear that such a single-enzyme view does not reflect the i n v i v o , where a actual situation variety of genetic and epigenetic factors may affect the basic steps of melanogenesis. A number of enzymes, particularly those of the glutathione system, have been shown to play a critical role in the regulation of the amount and type of pigment formed (2-4), while others, like thioredoxin reductase ( 5 ) , superoxide dismutase (6,7), and dopachrome oxidoreductase (8,9) or dopachrome tautomerase (10) have been postulated to affect more or less directly the process of melanogenesis on the whole. In the early 1970’s Okun and coworkers first proposed that peroxidase rather than, or possibly, in addition to, tyrosinase could catalyze the oxidative conversion of tyrosine to melanin. These authors obtained histochemical and biochemical evidence suggesting that oxidation of tyrosine to melanin in a variety of cells, including eosinophils, mast cells,
melanocytes and neurons could be mediated by a peroxidase (11). Further support to a role of peroxidase in melanogenesis came from the i n v i t r o demonstration of the formation of dopachrome by peroxidatic oxidation of tyrosine (12,13). On these grounds, Okun concluded that npmammalian peroxidase can mediate the conversion of tyrosine to melanin in the presence of dopa as cofactor as well as the conversion of dopa to melanin.pn and moreover that In there is no evidence that mammalian tyrosinase has significant ability to mediate the conversion of tyrosine to melanin, though it has strong dopa oxidase activitypn. Iltyrosinase may, therefore, act on dopa formed by peroxidase catalysisnn(14). Such an interpretation of the enzymatic assistance in melanogenesis did not pass unnoticed and stimulated considerable activity in various laboratories. Thus, a few years later, Swan repeated the i n v i t r o experiments of Okun and concluded that “NO evidence was found that tyrosine was hydroxylated by peroxidase in the presence of hydrogen peroxide and dopa According to as cofactor. In (15) Swan, Okunls report of dopachrome formation in the incubation mixture containing tyrosine, dopa and peroxidase-hydrogen peroxide was actually a misinterpretation of the spectral changes occurring during the reaction. Further support to this view came from the work of Hearing and Eke1 (16) providing evidence that a true tyrosinase is present in mammalian melanocytes. This series of papers apparently settled the question of peroxidase as an alternative to
..
..
.
G . Prota
26
tyrosinase and re-assured the supporters of tyrosinase-centered theories of melanogenesis, thus relegating the peroxidase hypothesis to history. Recently, however, the story of peroxidase was taken up again in our laboratory, when we began a systematic investigation of the role of this enzyme in melanogenesis. Emphasis in this context was directed at comparing the ability of peroxidase v s tyrosinase in catalyzing the later stages of the biosynthetic process involving the oxidative polymerization of 5,6-dihydroxyindole (DI) and 5,6-dihydroxyindole-2-carboxylic acid (DICA) A brief account of the results which emerged from these studies (17,18) is given in the following.
.
VS. TYROSINASE OXIDATION OF DI
PEROXIDASE
PROMOTED
In preliminary experiments , the course of the oxidation of DI by mushroom tyrosinase or horseradish peroxidase in the presence of hydrogen peroxide was investigated spectrophotometrically. With both enzymes, the reaction led to the formation of a purple pigment with a broad maximum at about 540 nm, closely reminiscent of melanochrome (19,20). Inspection of the W region of the spectrum revealed for the tyrosinase catalyzed reaction the persistence of the characteristic DI absorption bands at 272 and 298 (21) nm for at least the first few minutes whereas, in the case of peroxidase, the initial DI chromophore immediately coalesced into the developing W absorption band of the pigment product, covering approximately the same wavelength range. HPLC determination of DI concentration as a function of time (fig. 1) in the tyrosinase (I) and peroxidase (11) promoted oxidations, under the above experimental conditions, clearly indicated that peroxidase is by far more effective than tyrosinase in causing the fast and coniplete oxidation of the substrate: however soon the mixture was analyzed for DI after addition of peroxidase no detectable amount of residual indole could be observed, whereas, in the presence of tyrosinase, 50 % depletion of DI was reached not earlier than 40 seconds, with an initial rate of 4.4+ 0.2 xlO-' M / s . That the fast decompo
Time (rnin)
Fig.1. Kinelic course of the oxidalion of D1 ( 3 . 0 ~ 1 0 M) - ~ in 0.025 M phosphate buffer, pH 6.8. by I) mushfoom tyrosinase ( 2 . 7 ~ 1 0 - ~ U/ml) or 11) horseradish peroxidase (0.44 pyrogallol U/ml)-hydrogen peroxide (l.ZxlO-' M) as followed by HPLC moniloring of substrate decay. Aliquots of the reclion mixlures were periodically wilhdrawn. poured into test tubes containing solid ascorbic acid to stop lhe reaction and then analyzed by HPLC.
sition of DI in the presence of peroxidase was not merely due to a non-enzymic oxidation of the indole caused by the hydrogen peroxide cosubstrate was apparent from separate control experiments, showing that under the conditions of the peroxidase assay, but without added enzyme, only a 20% consumption of the substrate occurs after 60 min, on account of its intrinsic instability. Another major difference in the tyrosinase and peroxidase oxidation of DI was in the pattern of products formed. With both enzymes, the direct analysis of the purplish materials formed proved impracticable, owing to their limited solubility and unfavorable chromatographic properties. Accordingly, a standard work up procedure (22), involving dithionite reduction of the oxidation mixture, followed by acetylation of the ethyl acetate extractable fraction, was adopted. The typical HPLC elution profiles obtained under these conditions are shown in fig. 2. The upper chromatogram (I), which refers to the tyrosinase catalyzed reaction as stopped after 20 min, provided evidence for a very complex and spread pattern of products, among which only one major component ( A ) could clearly be distinguished along with the starting material as diacetyl derivative (DAI). The relevant peak could be isolated by preparative HPLC,
27
G . Rota
polymeric species, and the acetylated product pattern was comparatively less resolved. In the case of peroxidase, on the other hand, the product pattern (11) was considerably more defined, consisting, besides dimer 1 (peak A) , of another major component (B) and lower amounts of products C and D. Careful fractionaction by flash chromatography coupled with preparative HPLC allowed isolation of compounds corresponding to peaks B, C, and D which were formulated as the 2,7'-dimer 2 and the trimers 3 and 4 , respectively (fig.3) on the basis of their spectral features (17). The estimated yields for dimers 1 and 2 were 20-25% each and 3-5% for the isomeric trimers 3 and 4.
I
I
2
I
I
I
I
10
min
14
I
I
6
The identification of oligomers 1-4 is of great interest, as they can be regarded, with a rather pictorial definition, though perhaps not entirely correct , as Ilembryonic melanins" From consideration of their structures it appears that the dominant mode of coupling of DI is v i a the 2 and 4 positions of the heterocyclic ring, with a minor route v i a the 2 and 7 positions. It should be noted that such mode of polymerization is in contrast with the results of previous works carried out in the 1960Is showing that the 3- and 7-positions of the 5,6-dihydroxyindole nucleus were primarily involved in the formation of melanin pigment (23).
.
B
I1
A
DAI 0.01 A
C
-
TYROSINASE VS. PEROXIDASE OXIDATION OF DICA.
J I
I
I
I
I
I
I
2 6 10 min 14 Fig. 2. HPLC elution profile of the products formed by oxidation of DI (3.OxlO-' M) in 0.1 M phosphate buffernpH6.8 promoted by I) tyrosinase (4.9xlV3U/ml) or peroxidase (4.2~10"U/ml) /hydrogen peroxide (3.OxlO-' M). The reaction was stopped after 20 min in the case of tyrosinase and after 2 min for peroxidase and the mixture worked up according to Corradini et al. (22).
and the product identified as the 2 ,4-dimer 1 (fig.3) by spectral analysis (17). The estimated yield of dimer 1 fell in the 5-10% range with respect to DI for three separate experiments. Prolonging the reaction time up to 1 h resulted in further consumption of the substrate, but led to a substantially lower yield of extractable material, probably owing to the increased formation of insoluble
PROMOTED
In a second series of experiments, investigation was directed at accertaining the ability of the peroxidase/H202 system to effect the oxidation of DICA, the 5,6-dihydroxyindole compound resulting from the non decarboxylative rearrangement of dopachrome whose role in melanin biosynthesis has not still been definitely assessed. A comparison of the oxidation of DICA by tyrosinase or peroxidase showed differences in the kinetic and chemical course of the reaction even more striking than those observed with DI. By periodical monitoring of UV absorption scan curves evidence was obtained that tyrosinase was quite ineffective in bringing about the oxidation of the indole, whereas peroxidase was able to cause a significant reaction, leading in a
G . Prota
28
AcO
1
AcoqQ
AcO
3
2
q Ac
4
Fig. 3. Structures of the oligomers obtained by tyrosinase and peroxidase catalyzed oxidation of DI
100
X DlCl
80 60 40
2E
0
10
20
30
40
50
60
Time (rnin)
Fig. 4. Kinetic course of the oxidation of DICA (3.0~10-~ M) in 0.025 M phosphate buffer. pH 6.8. by 1) tyrosinase or 11) peroxidase/hydrogen peroxide . Reaction conditions and analysis of the mixtures were identical to those described in fig. 1.
relatively brief time to a melanochrome-like pigment with absorption maximum at 540 nm. The marked difference in the rates of the tyrosinase and peroxidase promoted oxidation of DICA was apparent also from the HPLC determination of substrate decay against time (fig.4). The resulting data showed a very poor catalytic effect of tyrosinase (I) on the oxidation of DICA (initial rate of about 5.6k0.2 x10b6 M / s ) , in striking contrast with the behavior of peroxidase (11), which caused the
complete disappearance of the indole within the very first seconds of the reaction (10 or less). Direct HPLC analysis of the purple pigment formed by peroxidase promoted oxidation of DICA failed to provide any significant information on its chemical nature and composition. A well defined pattern of products (fig.5) was however obtained by a recently developed work up procedure (24) involving reduction of the pigment and sequential treatment of the acidic ethyl acetate extractable fraction with methanolic HC1, to convert carboxyl groups into their methyl esters, and acetic anhydride/pyridine. Besides small amounts of DICA in the form of diacetyl methylester (DAICA ester) , two main products (E,F) could be isolated from the reaction mixture, which, on analysis, proved identical with the 4,4'-dimer 5 and the trimer 6 (fig.6), previously obtained by enzymic and metal catalyzed oxidation of DICA (24). Average yields of 5 and 6 were 10% and 25%, respectively. The structures of dimer 5 and trimer 6, both of which contain a symmetrical 4,4'-biindolyl unit, taken together with those of DI oligomers, offer an interesting chemical background for our biosynthetic approach to the structure of melanin.
G.Prota
29
F
DAlCA
0.0 A
2
14
la min
6
Fig. 5. HPLC elution profile of the products formed by oxidation of DICA ( 5 . 0 ~lo4 M) promoted by peroxidase (7.3U/ml) hydrogen peroxide (50x10 M) in 0.1 M phosphate buffer, pH 6.8. After 15 rnin the reaction was stopped and the mixture worked up according to Palumbo et al. (24).
particular interest is the observed ability of peroxidase to catalyze the oxidative polymerization of DICA to melanin. This offers a plausible biochemical explanation to the reported high degree of incorporation of DICA into natural melanins, which would otherwise be difficult to account in terms of a tyrosinase promoted oxidation. The profound difference in the oxidizing abilities of tyrosinase and peroxidase towards DICA merits attention also in relation to the intriguing finding by Mishima and coworkers that in the coated vesicles of mammalian melanocytes tyrosinase and DICA coexist, yet no melanin is formed (25,26). Such an apparent discrepancy would in fact be reconciled by the low oxidizability of DICA by tyrosinase that emerged from this study. This would also imply that under conditions of active melanogenesis enzymic systems other than tyrosinase actually bring about the oxidation of DICA to melanin. According to our studies, peroxidase would be a plausible candidate. We believe that this enzyme has the potential to be a most potent vicarious in vivo, system of tyrosinase especially for what concerns the diffusible indole intermediates of melanogenesis, formed in the later stages of the process. Support for this view has recently been gained by a cooperative study we carried out with drs. Mishima and Shibata at Kobe University which showed that high levels of peroxidase activity are associated with premelanosomes and mature melanosomes of melanotic cells while they are lower in coated vesicles where no significant melanin formation was found to occur (27). Relevant to our studies is also a recent report by Moellman and Halaban (28) that the protein encoded by the brown b-locus of mouse chromosome 4 is not tyrosinase, (29), but a as previously held catalase.
Acopo& AcO
"
'
m 0O
&
AcO
5
H
3
H
6
Fig. 6. Structures of the oligomers obtained by peroxidase catalyzed oxidation of DICA
CONCLUSIONS. Overall, the results of our i n vitro studies provide evidence that peroxidase can efficiently catalyze three critical steps in the biosynthesis of melanins, that is the conversion of dopa to dopaquinone; the oxidative polymerization of DI; and the oxidative polymerization of DICA. The role of this enzyme in the later stages of melanogenesis has never been investigated before and opens new perspectives in the understanding of the complex regulatory mechanisms of skin melanin pigmentation. Of
All together, these results suggest that, in addition to tyrosinase, peroxidase and possibly catalase play an important role in melanin pigmentation.
ACKNOWLEDGMENTS This work was supported by grants from Minister0 Pubblica Istruzione (MPI 6 0 % , 40% , Rome) and tne Lawrence M. Gelb Research Foundation (Stamford, CT, USA)
G . Prota REFERENCES. E, Bertrand A.Le 1) Bourquelot bleuissement et le noircissement des champignons. C R Soc Biol 1895:47:582-4. 2) Prota G. Melanin and Pigmentation. In: Dolphin D, Paulson R, Abramovic 0. eds. Coenzymes and Cofactors. New York:John Wiley & Sons, 1988:3B:538-72. 3) Mojamdar M, Ichihashi M, Mishima Y. 7-Glutamyl transpeptidase, tyrosinase and 5-S-cysteinyldopa production in melanoma cells. I Invest Dermatol 1983:81:119-21. 4) Rorsman H, Agrup G, Hansson c, Rosengren E.Biochemica1 recorders of malignant melanoma .In: Mackie RM, ed. Pigment Cell. Basel:Karger, 1983:6:93-115. 5) Schallreuter KU, Wood JM. The role of thioredoxin reductase in the reduction of free radicals at the surface of the epidermis. Biochem Biophys Res Commun 1986:136:630-7. 6) Sichel G. Biosynthesis and function of melanins in hepatic pigmentary system. Pigment Cell Res. 1988:l:250-258.
7) Pathak MA. Photoprotective role of melanin (eumelanin) in human skin. In: Douglas RH, Moan J, Dall'Acqua F eds. Light in Biology and Medicine. New York:Plenum Press, 1987:337-344. 8) Pawelek J, Korner A, Bergstrom A, Bologna J.New regulators of melanin biosynthesis and the autodestruction of melanoma cells.Nature 1980:286:617-9. 9) Barber JI, Townsend DW, Olds DP, King RA. Dopachrome oxidoreductase: a new enzyme in the pigment pathway. J Invest Dermatol 1984:83:145-9. 10) Aroca P, Garcia Borron JC, Solano F, Lozano J A . Regulation of mammalian melanogenesis. 1:partial purification and characterization of a dopachrome converting factor: dopachrome tautomerase. Biochim. Biophys. Acta 1990:1035:266-275.. 11) Okun M, Edelstein LM, Hamada G, Donnellan B. The role of peroxidase vs. the role of tyrosinase in the enzymatic conversion of tyrosine to melanin in melanocytes, mast cell and eosinophils; an autoradiographic-histochemical study. J Invest Dermatol 1970:55:1-12. 12) Patel RP, Okun MR, Edelstein LM, Cariglia N. Peroxidatic conversion of tyrosine to dopachrome. J Invest Dermatol 1974:63:374-7. 13) Patel RP, Okun MR, Edelstein LM, Epstein D. Biochemical studies on the peroxidase-mediated oxidation of tyrosine to melanin: demonstration of the hydroxylation of tyrosine by plant
and human peroxidases. Biochem J 1971:124:439-41. 14) Okun MR, Edelstein LM, Patel RP, Donnellan B. A revised concept of mammalian melanogenesis: the possible synergistic functions of aerobic dopa oxidase and peroxidase. A review. Yale J Biol Med 1973: 46:535-40. 15) Smith PI, Swan GA. A study of the supposed hydroxylation of tyrosine catalysed by peroxidase Biochem J 1976: 153:403-8. 16) Hearing VJ, Eke1 TM. Involvement of tyrosinase in melanin formation in murine melanoma. J Invest Dermatol 1975: 64:80-3. 17) d'Ischia M, Napolitano A, Tsiakas K. Prota G. New intermediates in the oxidative polymerisation of 5,6-dihydroxyindole to melanin promoted by the peroxidase/H202 system. Tetrahedron 1990: 46:5789-96. 18) d'Ischia M, Napolitano A, Prota G. Peroxidase as an alternative to tyrosinase in the oxidative polymerization of 5,6-dihydroxyindoles to melanin (s) Biochim Biophys Acta 1991, in press. 19) Mason HS. The chemistry of melanin.. Mechanism of the oxidation of dihydroxyphenylalanine by tyrosinase. J Biol Chem 1948:172:83-92. 20) Beer RJS, Broadhurst T, Robertson A. The chemistry of melanins. Part V. The autoxidation of 5,6-dihydroxyindoles. J Chem SOC 1954: 1947-53. 21) Benigni JD, Minnis RL. The syntheses of 5,6-dihydroxyindole and some of its derivatives. J Heterocycl Chem 1965:2:387-392. 22) Corradini MG, Napolitano A, Prota G. A biosynthetic approach to the structure of eumelanins. The isolation of oligomers from 5,6-dihydroxy1-methylindole. Tetrahedron 1986:42:-
.
.
2083-88. 23) Swan GA. Structure, chemistry and
biosynthesis of the melanin. In: Herz W, Grisebach H, Kirby GW eds. Fortschritte der Chemie Organischer Naturstoffe. New York: Springer-Verlag, 1974:31:522-28. 24) Palumbo P, d'Ischia M, Prota G. Tyrosinase promoted oxidation of 5,6-dihydroxyindole-2-carboxylic acid to melanin. Isolation and characterization of oligomer intermediates. Tetrahedron 1987:43:4203-6. 25) Imokawa G, Mishima Y. Isolation and biochemical characterization of tyrosinase-rich GERL and coated vesicles in the melanin synthetizing cell. Br J Dermatol 1981:104:169-78. 26) Chakraborty AK, Mishima Y, InaZU MI Hatta S, Ichihashi M. Melanogenic
G . F’rota regulatory factors in coated vesicles from melanoma cells. J Invest Dermatol 1989:93:616-9. 27) Shibata T I Prota G I Mishima Y. Regulatory factors of melanin monomer and polymer formation in melanogenic subcompartments of pigment cells. Abs CS-C8 XIV International Pigment Cell Conference, Kobe, Japan, 199O,p.91. 28) Halaban R, Moellmann G. Murine and human b locus pigmentation gene encode a glycoprotein (gp 75) with a catalase activity.Proc Natl Acad Sci USA 1990:87:4809-4813. 29) Hearing VJ, Himenez H. Analysis of mammalian pigmentation at the molecular level. Pigment Cell Res 1989:2:75-85.
31
The Role of Peroxidase in Melanogenesis Revisited GIUSEPPE PROTA Department of Organic and Biological Chemistry, University of Naples, Via Mezzocannone 16, 1-80134 Naples, Italy
Historically, our concept of melanogenesis developed from the early observation of Bourquelot and Bertrand (1) that tyrosinase was able to effect the oxidative conversion of tyrosine to a black insoluble pigment closely resembling natural melanins. This and in v i tro studies led subsequent gradually to a view of melanogenesis as a biochemically simple process involving the sole interaction of tyrosinase with the primary substrate tyrosine. However, as knowledge of the chemistry of melanin pigmentation has improved in more recent years, it has become increasingly clear that such a single-enzyme view does not reflect the i n v i v o , where a actual situation variety of genetic and epigenetic factors may affect the basic steps of melanogenesis. A number of enzymes, particularly those of the glutathione system, have been shown to play a critical role in the regulation of the amount and type of pigment formed (2-4), while others, like thioredoxin reductase ( 5 ) , superoxide dismutase (6,7), and dopachrome oxidoreductase (8,9) or dopachrome tautomerase (10) have been postulated to affect more or less directly the process of melanogenesis on the whole. In the early 1970’s Okun and coworkers first proposed that peroxidase rather than, or possibly, in addition to, tyrosinase could catalyze the oxidative conversion of tyrosine to melanin. These authors obtained histochemical and biochemical evidence suggesting that oxidation of tyrosine to melanin in a variety of cells, including eosinophils, mast cells,
melanocytes and neurons could be mediated by a peroxidase (11). Further support to a role of peroxidase in melanogenesis came from the i n v i t r o demonstration of the formation of dopachrome by peroxidatic oxidation of tyrosine (12,13). On these grounds, Okun concluded that npmammalian peroxidase can mediate the conversion of tyrosine to melanin in the presence of dopa as cofactor as well as the conversion of dopa to melanin.pn and moreover that In there is no evidence that mammalian tyrosinase has significant ability to mediate the conversion of tyrosine to melanin, though it has strong dopa oxidase activitypn. Iltyrosinase may, therefore, act on dopa formed by peroxidase catalysisnn(14). Such an interpretation of the enzymatic assistance in melanogenesis did not pass unnoticed and stimulated considerable activity in various laboratories. Thus, a few years later, Swan repeated the i n v i t r o experiments of Okun and concluded that “NO evidence was found that tyrosine was hydroxylated by peroxidase in the presence of hydrogen peroxide and dopa According to as cofactor. In (15) Swan, Okunls report of dopachrome formation in the incubation mixture containing tyrosine, dopa and peroxidase-hydrogen peroxide was actually a misinterpretation of the spectral changes occurring during the reaction. Further support to this view came from the work of Hearing and Eke1 (16) providing evidence that a true tyrosinase is present in mammalian melanocytes. This series of papers apparently settled the question of peroxidase as an alternative to
..
..
.
G . Prota
26
tyrosinase and re-assured the supporters of tyrosinase-centered theories of melanogenesis, thus relegating the peroxidase hypothesis to history. Recently, however, the story of peroxidase was taken up again in our laboratory, when we began a systematic investigation of the role of this enzyme in melanogenesis. Emphasis in this context was directed at comparing the ability of peroxidase v s tyrosinase in catalyzing the later stages of the biosynthetic process involving the oxidative polymerization of 5,6-dihydroxyindole (DI) and 5,6-dihydroxyindole-2-carboxylic acid (DICA) A brief account of the results which emerged from these studies (17,18) is given in the following.
.
VS. TYROSINASE OXIDATION OF DI
PEROXIDASE
PROMOTED
In preliminary experiments , the course of the oxidation of DI by mushroom tyrosinase or horseradish peroxidase in the presence of hydrogen peroxide was investigated spectrophotometrically. With both enzymes, the reaction led to the formation of a purple pigment with a broad maximum at about 540 nm, closely reminiscent of melanochrome (19,20). Inspection of the W region of the spectrum revealed for the tyrosinase catalyzed reaction the persistence of the characteristic DI absorption bands at 272 and 298 (21) nm for at least the first few minutes whereas, in the case of peroxidase, the initial DI chromophore immediately coalesced into the developing W absorption band of the pigment product, covering approximately the same wavelength range. HPLC determination of DI concentration as a function of time (fig. 1) in the tyrosinase (I) and peroxidase (11) promoted oxidations, under the above experimental conditions, clearly indicated that peroxidase is by far more effective than tyrosinase in causing the fast and coniplete oxidation of the substrate: however soon the mixture was analyzed for DI after addition of peroxidase no detectable amount of residual indole could be observed, whereas, in the presence of tyrosinase, 50 % depletion of DI was reached not earlier than 40 seconds, with an initial rate of 4.4+ 0.2 xlO-' M / s . That the fast decompo
Time (rnin)
Fig.1. Kinelic course of the oxidalion of D1 ( 3 . 0 ~ 1 0 M) - ~ in 0.025 M phosphate buffer, pH 6.8. by I) mushfoom tyrosinase ( 2 . 7 ~ 1 0 - ~ U/ml) or 11) horseradish peroxidase (0.44 pyrogallol U/ml)-hydrogen peroxide (l.ZxlO-' M) as followed by HPLC moniloring of substrate decay. Aliquots of the reclion mixlures were periodically wilhdrawn. poured into test tubes containing solid ascorbic acid to stop lhe reaction and then analyzed by HPLC.
sition of DI in the presence of peroxidase was not merely due to a non-enzymic oxidation of the indole caused by the hydrogen peroxide cosubstrate was apparent from separate control experiments, showing that under the conditions of the peroxidase assay, but without added enzyme, only a 20% consumption of the substrate occurs after 60 min, on account of its intrinsic instability. Another major difference in the tyrosinase and peroxidase oxidation of DI was in the pattern of products formed. With both enzymes, the direct analysis of the purplish materials formed proved impracticable, owing to their limited solubility and unfavorable chromatographic properties. Accordingly, a standard work up procedure (22), involving dithionite reduction of the oxidation mixture, followed by acetylation of the ethyl acetate extractable fraction, was adopted. The typical HPLC elution profiles obtained under these conditions are shown in fig. 2. The upper chromatogram (I), which refers to the tyrosinase catalyzed reaction as stopped after 20 min, provided evidence for a very complex and spread pattern of products, among which only one major component ( A ) could clearly be distinguished along with the starting material as diacetyl derivative (DAI). The relevant peak could be isolated by preparative HPLC,
27
G . Rota
polymeric species, and the acetylated product pattern was comparatively less resolved. In the case of peroxidase, on the other hand, the product pattern (11) was considerably more defined, consisting, besides dimer 1 (peak A) , of another major component (B) and lower amounts of products C and D. Careful fractionaction by flash chromatography coupled with preparative HPLC allowed isolation of compounds corresponding to peaks B, C, and D which were formulated as the 2,7'-dimer 2 and the trimers 3 and 4 , respectively (fig.3) on the basis of their spectral features (17). The estimated yields for dimers 1 and 2 were 20-25% each and 3-5% for the isomeric trimers 3 and 4.
I
I
2
I
I
I
I
10
min
14
I
I
6
The identification of oligomers 1-4 is of great interest, as they can be regarded, with a rather pictorial definition, though perhaps not entirely correct , as Ilembryonic melanins" From consideration of their structures it appears that the dominant mode of coupling of DI is v i a the 2 and 4 positions of the heterocyclic ring, with a minor route v i a the 2 and 7 positions. It should be noted that such mode of polymerization is in contrast with the results of previous works carried out in the 1960Is showing that the 3- and 7-positions of the 5,6-dihydroxyindole nucleus were primarily involved in the formation of melanin pigment (23).
.
B
I1
A
DAI 0.01 A
C
-
TYROSINASE VS. PEROXIDASE OXIDATION OF DICA.
J I
I
I
I
I
I
I
2 6 10 min 14 Fig. 2. HPLC elution profile of the products formed by oxidation of DI (3.OxlO-' M) in 0.1 M phosphate buffernpH6.8 promoted by I) tyrosinase (4.9xlV3U/ml) or peroxidase (4.2~10"U/ml) /hydrogen peroxide (3.OxlO-' M). The reaction was stopped after 20 min in the case of tyrosinase and after 2 min for peroxidase and the mixture worked up according to Corradini et al. (22).
and the product identified as the 2 ,4-dimer 1 (fig.3) by spectral analysis (17). The estimated yield of dimer 1 fell in the 5-10% range with respect to DI for three separate experiments. Prolonging the reaction time up to 1 h resulted in further consumption of the substrate, but led to a substantially lower yield of extractable material, probably owing to the increased formation of insoluble
PROMOTED
In a second series of experiments, investigation was directed at accertaining the ability of the peroxidase/H202 system to effect the oxidation of DICA, the 5,6-dihydroxyindole compound resulting from the non decarboxylative rearrangement of dopachrome whose role in melanin biosynthesis has not still been definitely assessed. A comparison of the oxidation of DICA by tyrosinase or peroxidase showed differences in the kinetic and chemical course of the reaction even more striking than those observed with DI. By periodical monitoring of UV absorption scan curves evidence was obtained that tyrosinase was quite ineffective in bringing about the oxidation of the indole, whereas peroxidase was able to cause a significant reaction, leading in a
G . Prota
28
AcO
1
AcoqQ
AcO
3
2
q Ac
4
Fig. 3. Structures of the oligomers obtained by tyrosinase and peroxidase catalyzed oxidation of DI
100
X DlCl
80 60 40
2E
0
10
20
30
40
50
60
Time (rnin)
Fig. 4. Kinetic course of the oxidation of DICA (3.0~10-~ M) in 0.025 M phosphate buffer. pH 6.8. by 1) tyrosinase or 11) peroxidase/hydrogen peroxide . Reaction conditions and analysis of the mixtures were identical to those described in fig. 1.
relatively brief time to a melanochrome-like pigment with absorption maximum at 540 nm. The marked difference in the rates of the tyrosinase and peroxidase promoted oxidation of DICA was apparent also from the HPLC determination of substrate decay against time (fig.4). The resulting data showed a very poor catalytic effect of tyrosinase (I) on the oxidation of DICA (initial rate of about 5.6k0.2 x10b6 M / s ) , in striking contrast with the behavior of peroxidase (11), which caused the
complete disappearance of the indole within the very first seconds of the reaction (10 or less). Direct HPLC analysis of the purple pigment formed by peroxidase promoted oxidation of DICA failed to provide any significant information on its chemical nature and composition. A well defined pattern of products (fig.5) was however obtained by a recently developed work up procedure (24) involving reduction of the pigment and sequential treatment of the acidic ethyl acetate extractable fraction with methanolic HC1, to convert carboxyl groups into their methyl esters, and acetic anhydride/pyridine. Besides small amounts of DICA in the form of diacetyl methylester (DAICA ester) , two main products (E,F) could be isolated from the reaction mixture, which, on analysis, proved identical with the 4,4'-dimer 5 and the trimer 6 (fig.6), previously obtained by enzymic and metal catalyzed oxidation of DICA (24). Average yields of 5 and 6 were 10% and 25%, respectively. The structures of dimer 5 and trimer 6, both of which contain a symmetrical 4,4'-biindolyl unit, taken together with those of DI oligomers, offer an interesting chemical background for our biosynthetic approach to the structure of melanin.
G.Prota
29
F
DAlCA
0.0 A
2
14
la min
6
Fig. 5. HPLC elution profile of the products formed by oxidation of DICA ( 5 . 0 ~lo4 M) promoted by peroxidase (7.3U/ml) hydrogen peroxide (50x10 M) in 0.1 M phosphate buffer, pH 6.8. After 15 rnin the reaction was stopped and the mixture worked up according to Palumbo et al. (24).
particular interest is the observed ability of peroxidase to catalyze the oxidative polymerization of DICA to melanin. This offers a plausible biochemical explanation to the reported high degree of incorporation of DICA into natural melanins, which would otherwise be difficult to account in terms of a tyrosinase promoted oxidation. The profound difference in the oxidizing abilities of tyrosinase and peroxidase towards DICA merits attention also in relation to the intriguing finding by Mishima and coworkers that in the coated vesicles of mammalian melanocytes tyrosinase and DICA coexist, yet no melanin is formed (25,26). Such an apparent discrepancy would in fact be reconciled by the low oxidizability of DICA by tyrosinase that emerged from this study. This would also imply that under conditions of active melanogenesis enzymic systems other than tyrosinase actually bring about the oxidation of DICA to melanin. According to our studies, peroxidase would be a plausible candidate. We believe that this enzyme has the potential to be a most potent vicarious in vivo, system of tyrosinase especially for what concerns the diffusible indole intermediates of melanogenesis, formed in the later stages of the process. Support for this view has recently been gained by a cooperative study we carried out with drs. Mishima and Shibata at Kobe University which showed that high levels of peroxidase activity are associated with premelanosomes and mature melanosomes of melanotic cells while they are lower in coated vesicles where no significant melanin formation was found to occur (27). Relevant to our studies is also a recent report by Moellman and Halaban (28) that the protein encoded by the brown b-locus of mouse chromosome 4 is not tyrosinase, (29), but a as previously held catalase.
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'
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Fig. 6. Structures of the oligomers obtained by peroxidase catalyzed oxidation of DICA
CONCLUSIONS. Overall, the results of our i n vitro studies provide evidence that peroxidase can efficiently catalyze three critical steps in the biosynthesis of melanins, that is the conversion of dopa to dopaquinone; the oxidative polymerization of DI; and the oxidative polymerization of DICA. The role of this enzyme in the later stages of melanogenesis has never been investigated before and opens new perspectives in the understanding of the complex regulatory mechanisms of skin melanin pigmentation. Of
All together, these results suggest that, in addition to tyrosinase, peroxidase and possibly catalase play an important role in melanin pigmentation.
ACKNOWLEDGMENTS This work was supported by grants from Minister0 Pubblica Istruzione (MPI 6 0 % , 40% , Rome) and tne Lawrence M. Gelb Research Foundation (Stamford, CT, USA)
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