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Jan 24, 2018 - have overlapping absorbance spectra, it is possible that these rates contain contribution(s) from other intermediates besides. DC as we...
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Cite This: J. Phys. Chem. B 2018, 122, 2047−2063

Kinetics of Melanin Polymerization during Enzymatic and Nonenzymatic Oxidation Sayan Mondal, Arya Thampi, and Mrinalini Puranik* Indian Institute of Science Education and Research, Pune 411008, India S Supporting Information *

ABSTRACT: Melanin is an abundant biopigment in the animal kingdom, but its structure remains poorly understood. This is a substantial impediment to understanding the mechanistic origin of its observed functions. Proposed models of melanin structure include aggregates of both linear and macrocyclic units and noncovalently held monomers. Both models are broadly in agreement with current experimental data. To constrain the structural and kinetic models of melanin, experimental data of high resolution with chemical specificity accompanied by atomistic modeling are required. We have addressed this by obtaining electronic absorption, infrared, and ultraviolet resonance Raman (RR) spectra of melanin at several wavelengths of excitation that are sensitive to small changes in structure. From these experiments, we observed kinetics of the formation of different species en route to melanin polymerization. Exclusive chemical signatures of monomer 3,4-dihydroxyphenylalanine (dopa), intermediate dopachrome (DC), and early-time polymer are established through their vibrational bands at 1292, 1670, and 1616 cm−1 respectively. Direct evidence of reduced heterogeneity of melanin oligomers in tyrosinase-induced formation is provided from experimental measurements of vibrational bandwidths. Models made with density functional theory show that the linear homopolymeric structures of 5,6-dihydroxyindole can account for experimentally observed wavenumbers and broad bandwidth in Raman spectra of dopa-melanin. We capture resonance Raman (RR) signature of DC, the intermediate stabilized by the enzyme tyrosinase, for the first time in an enzyme-assisted melanization reaction using 488 nm excitation wavelength and propose that this wavelength can be used to probe reaction intermediates of melanin formation in solution.

1. INTRODUCTION Melanins refer to a broad class of heterogeneous biopolymers that are responsible for the natural color of the skin, hair, and eyes of mammals. Melanins are synthesized within specialized membrane-bound organelles known as melanosomes with the help of a copper-containing enzyme tyrosinase (TYR). There exist two major types of melanins: brown-to-black eumelanin and reddish-brown pheomelanin. The enzymatic synthesis pathway of eumelanin, which is also the focus of this article, is known as Raper−Mason scheme1−6 (Figure 1). Fundamental building blocks of eumelanins are two indolic molecules, 5,6-dihydroxyindole (DHI) and 5,6-dihydroxyindole-2-carboxylic acid (DHICA), both of which are oxidative products of an intermediate known as dopachrome (DC) (Figure 1). Structure and self-assembled organization of this ubiquitous pigment have been studied at different levels of structural hierarchy through both the top-down and bottom-up approaches from macro (microns) to nano (nanometer) length scales using experimental and theoretical methods and have been documented in several reviews.7−17 Initially, eumelanins were thought of as amorphous semiconductors,18−21 but muon spin relaxation and electron paramagnetic resonance experiments have established that © 2018 American Chemical Society

melanin has a hydration-state-dependent hybrid protonic− electronic conductivity.22,23 Melanin’s broad UV−visible (vis) absorbance has been traditionally explained with an extended heteropolymer model.24−27 The model proposes that melanin is a polymer formed via covalent bonding between any two available sites of indolic monomers and neglects further specific details of interunit coupling. Subsequently, the broad-band absorption, which is usually linked with its photoprotective role, has been explained as a superposition of a large number of Gaussian bands, each associated with a slightly different heteropolymer (5−6 monomer units that arrange in an extended sheetlike structure) by Stark et al.28−30 and Meredith and coworkers.31 The latter group termed it the chemical disorder model.10,31,32 Later, the finite-sized stacked sheet model emerged from X-ray diffraction experiments,33−36 scanning tunneling microscopy,37−39 and atomic force microscopy (AFM) imaging40−42 by several groups on synthetic and natural (from Sepia officinalis and referred as Sepia melanin from here on) pigments. These Received: August 27, 2017 Revised: January 7, 2018 Published: January 24, 2018 2047

DOI: 10.1021/acs.jpcb.7b07941 J. Phys. Chem. B 2018, 122, 2047−2063

Article

The Journal of Physical Chemistry B

Figure 1. Raper−Mason scheme of eumelanin production pathway using both tyrosine and L-3,4-dihydroxyphenylalanine (L-dopa) as precursor. Both dopa and tyrosine get oxidized in the presence of the enzyme tyrosinase (TYR) to form dopaquinone (DQ), which readily undergoes cyclization and forms dopachrome (DC) through the intermediate cyclodopa. 5,6-Dihydroxyindole (DHI) or 5,6-dihrdoxyindole-2-carboxylic acid (DHICA) is produced from DC via decarboxylation or spontaneous rearrangement, respectively. The production of DHICA only happens in the presence of an enzyme called dopachrome tautomerase (DCT, also known as TYRP2). The thus-formed DHI and DHICA get oxidized to form the pigment precursor melanochrome (MC) and subsequently melanin. Oxidation of DHICA is catalyzed by an enzyme known as DHICA oxidase (known as TYRP1 also). The possible polymerization sites are marked on structures of both DHI (second, fourth, and seventh) and DHICA (second and fourth). In the current study, we are investigating the pathway of DHI melanin formation from dopa via auto-oxidation and tyrosinase-assisted oxidation of dopa.

hand, linear protomolecules of melanin have been observed in experiments by Prota, d’Ischia, and co-workers.46−52 Solutionstate nuclear magnetic resonance (NMR) and mass spectroscopic analyses of the oxidative products of DHI and/or DHICA showed that initial polymeric species are small linear oligomers up to tetramers made from monomer units that are intertwisted with respect to each other. Early oligomerization primarily occurs through covalent bonding at 2, 4, and 7 positions in DHI47,50 (Figure 1) and 4 and 7 positions in DHICA49 (Figure 1). The most recent model of a synthetic melanin, polydopamine (dopamine-melanin), proposes that the pigment is not a covalently linked polymer but a supramolecular structure assembled by aggregation of monomers through charge transfer, π-stacking, and hydrogen-bonding interactions.53,54 Semiempirical and density functional theory (DFT) calculations on a variety of protomolecules, including macrocycles, finitely extended sheets, and linear oligomers, show that the enhanced UV−visible absorption at high-energy (600 min), monomer signature (1292 cm−1) is still observed as a result of remaining unused dopa. This can be as a result of complete depletion of oxygen (which was present as soluble portion in the enzyme solution at the beginning of reaction), thus making the remaining tyrosinase inactive. Recently, the use of excess amount of tyrosinase has been suggested during production of enzymeassisted melanin.14 Moreover, once DC is formed with the help of the amount of oxygen present initially, the consecutive downstream reactions get spontaneously triggered. The role of tyrosinase during these steps is not conclusively known. Deciphering the role of stoichiometry of tyrosinase, the precursor, and dissolved oxygen on observed reaction rates needs further investigations. To delineate these steps conclusively, rapid-mixing experiments that can simultaneously probe concentrations of these interconverting species are necessary. DC decays to DHI via spontaneous decarboxylation and subsequently forms a precursor of pigment, MC. The absence of spectroscopic signature of DHI in any of our measurement implies higher formation and decay rates than that could be detected in our experiment. Decay of DC and formation of MC is explained by considering, at least, two intermediates, IN−1 and IN. DC decays and IN−1th intermediate forms with rates of 5.2 × 10−2 and 1.4 × 10−2 min−1, respectively. Consecutively, the IN−1th intermediate converts to IN that subsequently produces MC with a lower rate of ∼9.1 × 10−3 min−1. Overall, in the presence of tyrosinase, the formation and decay rates of DC are found to be 1 order of magnitude higher than the consumption rate of dopa and the formation rates of IN and MC. In autoxidation, DC is not stable enough to be detected, and the overall kinetics follows the rates that are lower than those in tyrosinase-catalyzed reaction. Falguera et al. noted an interesting fact: a gradual decrease of extinction coefficient (ε) of DC at 480 nm with the increase in the initial concentration of the precursor.64 This is a consequence of the fact that in their kinetic model ε is kept a free parameter. The extinction coefficient is an intrinsic property of a molecule. These authors rationalize their observation as melanin formation is a complex reaction, in which a group of heterogeneous length chain pigments are formed. In this way, chains with different compositions will affect differently to the global coefficient. Our data clearly demonstrate that absorbance at 480 nm has a significant amount of contribution from early time pigment (melanochrome) and melanin itself. Thus, for the successful development of a kinetic model of melanin formation, DC and melanin have to be probed with noninterfering spectroscopic probes, such as distinct vibrational signature, as reported in the current study. Fluorescence of thioflavin T (ThT) was used by Sutter et al. to analyze the kinetics of dopa-melanin formation (through autoxidation) in solution phase.73 ThT is a molecular rotor that has weakly emissive state in solution but fluoresces once interunit rotation is constrained.119 They have observed an initial time lag, then rise, and finally saturation of ThT fluorescence during the course of melanization reaction. From this observation, it was inferred that as melanin starts aggregating

5. CONCLUSIONS We have examined the initial polymerization process of melanin with multiple probes, Raman, infrared, UV−visible absorption, and computational models. We have used vibrational spectroscopy to obtain chemical identity of the intermediates of melanin and used these to follow the kinetics of the reaction. Using a kinetic model, we report the rates of consumption of the monomeric melanin precursor, dopa, formation and decay of the key intermediate dopachrome, and formation of melanochrome. Molecule-specific vibrational markers allowed us to follow the fates of intermediates that have similar absorption spectra. Melanins produced in enzyme-assisted oxidation and autoxidation reactions are turned out to have vibrational fingerprint at the same 1615 cm−1 wavenumber position. However, we find that, tyrosinase not only increases rates of consumption of dopa and formation of melanin by an order of 2059

DOI: 10.1021/acs.jpcb.7b07941 J. Phys. Chem. B 2018, 122, 2047−2063

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The Journal of Physical Chemistry B

Catecholic Intermediate Substrate Responsible for the Autoactivation Kinetics of Tyrosinase. J. Biol. Chem. 1997, 272, 26226−26235. (7) Meredith, P.; Sarna, T. The Physical and Chemical Properties of Eumelanin. Pigm. Cell Res. 2006, 19, 572−594. (8) Ito, S. Reexamination of the Structure of Eumelanin. Biochim. Biophys. Acta, Gen. Subj. 1986, 883, 155−161. (9) Riley, P. A. Melanin. Int. J. Biochem. Cell Biol. 1997, 29, 1235−1239. (10) Meredith, P.; Powell, B. J.; Riesz, J.; Nighswander-Rempel, S. P.; Pederson, M. R.; Moore, E. G. Towards Structure−property−function Relationships for Eumelanin. Soft Matter 2006, 2, 37−44. (11) Simon, J. D.; Peles, D.; Wakamatsu, K.; Ito, S. Current Challenges in Understanding Melanogenesis: Bridging Chemistry, Biological Control, Morphology, and Function. Pigm. Cell Melanoma Res. 2009, 22, 563−579. (12) d’Ischia, M.; Napolitano, A.; Pezzella, A.; Meredith, P.; Sarna, T. Chemical and Structural Diversity in Eumelanins: Unexplored BioOptoelectronic Materials. Angew. Chem., Int. Ed. 2009, 48, 3914−3921. (13) Ito, S.; Wakamatsu, K.; D’ischia, M.; Napolitano, A.; Pezzella, A. Structure of Melanins. In Melanins and Melanosomes; Borovanský, J., Riley, P. A., Eds.; Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim, Germany, 2011; pp 167−185. (14) d’Ischia, M.; Wakamatsu, K.; Napolitano, A.; Briganti, S.; GarciaBorron, J.-C.; Kovacs, D.; Meredith, P.; Pezzella, A.; Picardo, M.; Sarna, T.; et al. Melanins and Melanogenesis: Methods, Standards, Protocols. Pigm. Cell Melanoma Res. 2013, 26, 616−633. (15) Solano, F. Melanins: Skin Pigments and Much MoreTypes, Structural Models, Biological Functions, and Formation Routes. New J. Sci. 2014, 2014, 1−28. (16) d’Ischia, M.; Napolitano, A.; Ball, V.; Chen, C.-T.; Buehler, M. J. Polydopamine and Eumelanin: From Structure−Property Relationships to a Unified Tailoring Strategy. Acc. Chem. Res. 2014, 47, 3541−3550. (17) d’Ischia, M.; Wakamatsu, K.; Cicoira, F.; Di Mauro, E.; GarciaBorron, J. C.; Commo, S.; Galván, I.; Ghanem, G.; Kenzo, K.; Meredith, P.; et al. Melanins and Melanogenesis: From Pigment Cells to Human Health and Technological Applications. Pigm. Cell Melanoma Res. 2015, 28, 520−544. (18) Longuet-Higgins, H. C. On the Origin of the Free Radical Property of Melanins. Arch. Biochem. Biophys. 1960, 86, 231−232. (19) Pullman, A.; Pullman, B. The Band Structure of Melanins. Biochim. Biophys. Acta 1961, 54, 384−385. (20) McGinness, J. E. Mobility Gaps: A Mechanism for Band Gaps in Melanins. Science 1972, 177, 896−897. (21) McGinness, J.; Corry, P.; Proctor, P. Amorphous Semiconductor Switching in Melanins. Science 1974, 183, 853−855. (22) Mostert, A. B.; Powell, B. J.; Pratt, F. L.; Hanson, G. R.; Sarna, T.; Gentle, I. R.; Meredith, P. Role of Semiconductivity and Ion Transport in the Electrical Conduction of Melanin. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 8943−8947. (23) Wünsche, J.; Deng, Y.; Kumar, P.; Di Mauro, E.; Josberger, E.; Sayago, J.; Pezzella, A.; Soavi, F.; Cicoira, F.; Rolandi, M.; et al. Protonic and Electronic Transport in Hydrated Thin Films of the Pigment Eumelanin. Chem. Mater. 2015, 27, 436−442. (24) Galvão, D. S.; Caldas, M. J. Polymerization of 5,6-Indolequinone: A View into the Band Structure of Melanins. J. Chem. Phys. 1988, 88, 4088. (25) Galvão, D. S.; Caldas, M. J. Theoretical Investigation of Model Polymers for Eumelanins. II. Isolated Defects. J. Chem. Phys. 1990, 93, 2848. (26) Galvão, D. S.; Caldas, M. J. Theoretical Investigation of Model Polymers for Eumelanins. I. Finite and Infinite Polymers. J. Chem. Phys. 1990, 92, 2630. (27) Bolívar-Marinez, L. E.; Galvão, D. S.; Caldas, M. J. Geometric and Spectroscopic Study of Some Molecules Related to Eumelanins. 1. Monomers. J. Phys. Chem. B 1999, 103, 2993−3000. (28) Stark, K. B.; Gallas, J. M.; Zajac, G. W.; Eisner, M.; Golab, J. T. Spectroscopic Study and Simulation from Recent Structural Models for Eumelanin: I. Monomer, Dimers. J. Phys. Chem. B 2003, 107, 3061− 3067.

magnitude compared to those in an autoxidation reaction but also drives the eumelanization through distinct pathway. The role of the enzyme is better understood and shows that the enzyme stabilizes selective intermediates and also controls the heterogeneity of melanin. Preceding the pigment formation, distinct population of short DHI polymers, implicated in several reports, is found to have smaller Raman bandwidth compared to their aggregated product, melanin. We explain the position and width of experimentally observed Raman band of melanin as an outcome of intrinsic structural heterogeneity due to a distribution of tetrameric oligomers. Our computational results show that the experimentally detected oligomeric scaffolds46−52 that are relatively unexplored as natural structural model of melanin need further investigations.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.7b07941. Resonance Raman data analysis procedure, geometric structures of lowest energetic DHI tetramers, and monomeric intermediates (DQ and cyclodopa); validation of used DFT method; vibrational assignment of dopa, DC, and substituted indoles; electronic structure and simulation of RR spectrum of DC; and computed potential energies of DHI tetramers (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +917350694600. Indian Institute of Science Education and Research, Dr. Homi Bhabha Road, Pashan, Pune 411008, India. ORCID

Sayan Mondal: 0000-0003-1051-8797 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by Indian Institute of Science Education and Research (IISER) Pune, Ministry of Human Resource Development, Government of India. S.M. gratefully acknowledges IISER Pune for a Ph.D. fellowship. A.T. would like to acknowledge INSPIRE scholarship from DST, Government of India for support during her Master thesis.

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REFERENCES

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