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Using size exclusion chromatography to monitor the synthesis of melanins from catecholamines
Accepted Manuscript Title: Using size exclusion chromatography to monitor the synthesis of melanins from catecholamines. Authors: Koen P. Vercruysse, Astiney M. Clark, Paola A.F. Bello, Majidah Alhumaidi PII: DOI: Reference:
S1570-0232(17)30142-3 http://dx.doi.org/doi:10.1016/j.jchromb.2017.04.005 CHROMB 20540
To appear in:
Journal of Chromatography B
Received date: Revised date: Accepted date:
25-1-2017 22-3-2017 1-4-2017
Please cite this article as: Koen P.Vercruysse, Astiney M.Clark, Paola A.F.Bello, Majidah Alhumaidi, Using size exclusion chromatography to monitor the synthesis of melanins from catecholamines., Journal of Chromatography Bhttp://dx.doi.org/10.1016/j.jchromb.2017.04.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Using size exclusion chromatography to monitor the synthesis of melanins from catecholamines.
Koen P. Vercruysse*,§, Astiney M. Clark*, Paola A. F. Bello*, Majidah Alhumaidi* *
Department of Chemistry, Tennessee State University; 3500 John A. Merritt Blvd., Nashville,
TN 37209
§
Corresponding author: e-mail: [email protected]; phone: +1-615-963-5340
Highlights
SEC is a versatile technique to monitor synthesis of melanins.
Many polysaccharides promote oxidation of catecholamines.
Polysaccharides form high molecular mass aggregates with melanins.
Abstract We have employed size exclusion chromatography (SEC) to the study of the auto- and Cu2+mediated oxidation of the catecholamines, dopamine, epinephrine and norepinephrine, into melanins. We observed that, due to non-size exclusion-mediated effects, the catecholamines and some of the low molecular mass intermediates generated during the oxidation reactions, could be resolved from each other and from the high molecular mass pigment generated. Thus, SEC
allowed us to monitor the disappearance of the catecholamine starting compounds, the appearance and subsequent disappearance of the low molecular mass chromophores generated in the initial phase of the reactions and the appearance of the high molecular mass melanins. In the process of this research, we observed that many, mostly anionic polysaccharides (PS), enhanced both the auto- and Cu2+-mediated oxidation of all three catecholamines. SEC analyses of reaction mixtures involving PS suggested that very high molecular mass aggregates between PS and melanins can be generated. In addition, SEC analysis allowed us to verify the efficiency of the dialysis purification process employed to obtain pure and dried melanin materials for cellbiological studies.
1. Introduction The pigments generated in our experiments belong to the category of the melanins (MN) and related biomolecules. MN are ubiquitously found in nature and excellent reviews regarding their biosynthesis, chemistry, classification and functions have been written.[1-3] The MN found in animals are built from L-DOPA which is derived from L-tyrosine. These types of MN are broadly divided into eumelanins and pheomelanins. The pheomelanins differ from the eumelanins that the amino acid L-cysteine is built into its structure. The pheomelanins have not yet been the subject of our experiments. The biosynthesis of MN involves a sequence of oxidation, cyclization and polymerization reactions most commonly described by the RaperMason scheme and illustrated in Figure 1.[1] In a first phase of this reaction scheme, the catechol portion of the precursor is converted into the o-quinone oxidation product and such o-quinones often exhibit absorbance in the visible region of the electromagnetic spectrum. Catecholamines (CAs) like epinephrine (EPI), norepinephrine (NE) or dopamine (DA) are biomolecules derived
from L-DOPA and can undergo a similar sequence of reactions leading to neuromelanins or other melanins, similar as for L-DOPA leading to eumelanins (see Figure 1). CAs like DA or NE are considered to be the precursors of the neuromelanins found in select areas of the brain.[4, 5] MN types of pigments are also produced by plants, fungi and bacteria, but the precursors of some of these pigments are often nitrogen-free phenols like catechol, caffeic acid, homogentisic acid or others.[3] Given the fact that MN constitute a broad range of biomolecules derived from a broad range of potential precursors, the qualitative and quantitative analysis and characterization is challenging, primarily due to the fact that the naturally-occurring MN are often insoluble.[6] Chromatographic approaches to the analysis of MN often involve the degradation of the pigment and the subsequent analysis of the degradation products.[6, 7] In this report we demonstrate the versatility of size exclusion chromatography (SEC) for the study of the formation of synthetic MN from CA precursors. In addition, we applied SEC to study the synthesis of MN from CAs in the presence of polysaccharides (PS). We observed that many PS promoted the oxidation reaction and that water-soluble, high molecular mass pigment/PS are generated in the process. Finally, we employed SEC to verify the dialysis purification process of the MN generated in our experiments.
2. Materials and Methods 2.1 Materials Chondroitin sulfate type A (sodium salt from bovine trachea; 70% with counterbalance chondroitin sulfate type C), chondroitin sulfate type C (sodium salt from shark cartilage; 90% with counterbalance chondroitin sulfate type A), alginic acid sodium salt (Algin®, sodium
alginate), -carrageenan (commercial grade, type II), carboxymethylcellulose (low viscosity grade; sodium salt), fucoidan (from Fucus vesiculosus, dopamine.HCl, epinephrine.HCl and norepinephrine.HCl, CuCl2.2H2O were obtained from Fisher Scientific (Suwanee, GA). All other reagents were of analytical grade. 2.3 UV/Vis spectroscopy UV/Vis spectra of DA, NE and EPI were obtained using a DU 800 spectrophotometer from Beckman Coulter (Fullerton, CA) against water as the blank. 2.4 RP-HPLC analyses RP-HPLC analyses were performed on a UFLC chromatography system equipped with dual LC6AD solvent delivery pumps and SPD-M20A diode array detector from Shimadzu, USA (Columbia, MD). Analyses were performed on a BDS Hypersil C8 column (125X4.6 mm) obtained from Fisher Scientific (Suwanee, GA). Analyses were performed in isocratic fashion using a mixture of water:methanol:acetic acid (90:10:0.05% v/v) as solvent. The sample volume was 20L. Samples were diluted to a CA concentration of approximately 0.1mM. 2.5 Size exclusion chromatography (SEC) SEC analyses were performed on a Breeze 2 HPLC system equipped with two 1500 series HPLC pumps and a model 2998 Photodiode array detector from Waters, Co (Milford, MA). Analyses were performed using an Ultrahydrogel 500 column (300 X 7.8 mm) obtained from Waters, Co (Milford, MA) in isocratic fashion using a mixture of 25mM Na acetate:methanol:acetic acid (90:10:0.05% v/v) as solvent. Samples were diluted with SEC solvent, centrifuged and 20 L was injected. Samples were diluted as indicated in the text or figures.
2.6 Dialysis and freeze drying Select samples were dialyzed using Spectrum Spectra/Por RC dialysis membranes with molecular-weight-cut-off of 3.5kDa obtained from Fisher Scientific (Suwanee, GA). Select dialyzed materials were frozen overnight and dried using a Labconco FreeZone Plus 4.5L benchtop freeze-dry system obtained from Fisher Scientific (Suwanee, GA). 2.7 FT-IR spectral analysis FT-IR spectroscopic scans were made using the NicoletiS10 instrument equipped with the SmartiTR Basic accessory from ThermoScientific (Waltham, MA). Scans were taken with a resolution of 4 cm-1 between 650 and 4,000 cm-1 at room temperature using a KBr beam splitter and DTGS KBr detector. Each spectrum represents the accumulation of 24 scans.
3. Results 3.1. Preliminary observations When Cu2+ was added to aqueous solutions containing EPI, DA or NE, a change in color, from colorless to pink, red or orange, appeared within hours of mixing depending on the concentrations of Cu2+ or CA involved. Figure 2 illustrates the UV_Vis spectra of the chromophores thus generated. For all three compounds the UV_Vis spectrum exhibited a distinct absorbance maximum between 450 and 500nm. It was presumed that these chromophores generated in the initial phase of the reaction corresponded to the o-quinone oxidation products of the CA precursors. When select PS were added to such reaction mixtures then within hours, e.g., fucoidan, or after overnight reaction, e.g., chondroitin sulfate (CS) A or C, the colors of the mixtures darkened to yellow-brown (EPI and NE) or grey-black (DA) (results not shown). These
observations prompted us to speculate that the chromophore generated initially reacted further, generating other types of colored substances. The reaction of CAs in the presence of Cu2+, with or without PS, was evaluated using RP-HPLC analyses. Figure 3 shows a typical RP-HPLC profile of EPI after reaction with Cu2+ in the presence or absence of CS A. EPI had a retention time of about 6 minutes and following reaction with Cu2+ new peaks emerged with retention times between 1 and 2.5 minutes that were poorly resolved. One peak within the 1 to 2.5 minute cluster had a UV_Vis spectrum similar to the ones shown in Figure 2 and this peak was presumed to be the chromophore generated in the initial phase of the reaction. RP-HPLC analyses of similar reactions that included select PS showed additional peaks, with retention times around 1 minute, as shown in Figure 3. RP-HPLC did not provide any resolution between the PS and the various reaction products generated from the CAs. However, when performing reactions in the presence of PS, the peak in the RP-HPLC profile associated with the chromophore declined or disappeared when the reactions were monitored over longer reaction times at RT (results not shown). In addition, the pattern of peaks observed in the 1 to 2.5 minute cluster changed significantly when monitored over longer periods of time (one day or longer). As before, it was speculated that the chromophore generated in the initial phase, reacted further, generating other types of colored substances. For the study of the oxidation of EPI, NE or DA in the presence of PS, RP-HPLC can be used to monitor the disappearance of the starting molecule under various reaction conditions, but can not adequately be used to evaluate the appearance of the various reaction products. 3.2. SEC studies The oxidation reactions of CAs and subsequent formation of pigmented materials was extensively evaluated using SEC. Reaction mixtures containing 0.5mM EPI, 0.03mM Cu2+ and 0
or 2.5 mg/L CS C were kept at RT for one week and, following two-fold dilution with SEC solvent, analyzed. Figure 4, panels A (signal at 275nm) and B (signal at 400nm), compares the SEC profiles of these reactions. For discussion purposes we subdivided the SEC profiles into four different regions. Region I includes peak retention times between 5 and 7 minutes; peak retention times thought to be close to the upper exclusion limit of the column. Region II includes peak retention times between 7 and 13 minutes; peak retention times typically observed following the injection of PS solutions and associated with the size distribution profiles of the PS. Region III includes peak retention times between 13 and 15 minutes; peak retention times close to the lower exclusion limit of the column as determined through the injection of water or other small molecules. Region IV includes peak retention times above 15 minutes; peak retention times typical for molecules with retentions determined by adsorption phenomena in addition to size-exclusion phenomena. In Figure 4, panel A, the peak with retention time of about 20 minutes corresponds to EPI. We observed that the SEC peak retention times of all three CAs fell in region IV of the SEC profiles (typically between 18 and 22 minutes). In Figure 4, panel A, the peak with retention time of about 16 minutes exhibited an UV_Vis profile similar to the one shown in Figure 2 (dotted line). It was presumed that this peak corresponds to the chromophore generated in the initial phase of the reaction. The SEC profiles of the reaction mixtures for all three CAs showed peaks with retention times between 15 and 17 minutes with UV_Vis profiles similar to the ones shown in Figure 2. When EPI or any of the other CAs reacted with Cu2+ in the absence of any PS, a cluster of up to three resolved peaks were observed in region III of the SEC profiles (see Figure 4, panel A). These peaks did exhibit absorbance in the visible region of the spectrum as shown in Figure 4, panel B. No peaks were observed in regions I or II of the SEC profiles. In the presence of PS, in addition to the peaks in regions III and IV of the SEC profiles,
broad peaks were observed in region II and, depending on the type of PS and CA involved, sharp peaks were sometimes observed in region I of the SEC profiles. The peaks in regions I or II of these SEC profiles exhibited absorbance in the visible region of the spectrum as shown in Figure 4, panel B. For a reaction involving 2.5 mg/mL CS C, 0.1mM Cu2+ and 1mM DA, SEC analyses were performed on diluted aliquots from the reaction mixture taken at various time intervals after the start of the reaction. Figure 5 shows the area-under-the-curve (AUC) of: (a) the peak corresponding to DA (retention time about 20min; using signal at 275nm), (b) the peak presumed to be corresponding to the o-quinone chromophore generated from DA (retention time about 18min; using signal at 300nm) and (c) the peak corresponding to CS C (retention time about 11.5min; using signal at 275nm). Figure 5 shows a rapid decline in the AUC corresponding to DA in the initial phase of the reaction, followed by a much slower decline. The AUC corresponding to the chromophore generated from DA increased in the initial phase of the reaction and then declined significantly. The AUC corresponding to CS C steadily increased as a function of the reaction time. Large scale reactions were set up that enabled us to isolate, purify and characterize as much as possible the pigmented materials generated through the reaction between select PS and CA without the addition of Cu2+. Figure 6 provides a picture of one such experiment. In a well of a plastic cell culture dish, about 30mg DA was dissolved in 10mL water. About 100mg test substance, carrageenan, alginate, CS A, CS C or fucoidan, was added as a powder and the mixtures were left at RT. Within minutes (carrageenan or fucoidan) or hours (alginate, CS A or CS C) dark colors appeared in these mixtures. The mixtures were left at RT for one week. At that point the picture shown in Figure 6 was taken and the pigments were siphoned off for dialysis
and freeze drying. Figure 7, panel A presents the SEC profile, shown at 230nm and 400nm, of the dialyzed reaction mixture involving DA and fucoidan as discussed for Figure 6. Two main peaks can be observed, one with a retention time of about 6.7 minutes and one with a retention time of about 8.9 minutes. When fucoidan is analyzed by itself, the SEC profile exhibits a single peak with a retention time of about 9 minutes (results not shown). Figure 7, panel B, presents the UV_Vis spectra of both peaks. Both peaks exhibit a similar pattern: strong absorbance in the UV region with an exponential decline in absorbance in the visible region. No distinct absorbance maxima could be observed. When fucoidan is analyzed, its UV_Vis spectrum displays absorbance in the UV region and none in the visible region (results not shown). Upon freeze drying, the material obtained from the reaction between fucoidan and DA consisted of black fibrous material and Figure 8 compares the FT-IR spectra of fucoidan and this dried material.
Discussion Our visual observations and UV_Vis, RP-HPLC or SEC analyses discussed for figures 2 through 5 regarding the oxidation of CAs and the corresponding changes in color are in line with the Raper-Mason scheme (see Figure 1) for the oxidation of DOPA to low molecular mass intermediates and their subsequent conversion into high molecular mass pigment. UV_Vis spectroscopy can be used to monitor the formation of colored substances in the oxidation reactions of the CAs, but does not allow one to make a clear distinction between the low molecular mass chromophores and the high molecular mass MNs present in the reaction mixtures. As shown in this report, RP-HPLC can be used to monitor the presence and decline of the CA starting compound in the oxidation reactions as the peaks corresponding to these molecules are well resolved from the other peaks in the chromatogram. However, the peaks
corresponding to the low molecular mass chromophores and other reaction products, including the high molecular mass MNs, are not resolved as they exhibit very low retention times. SEC provided sufficient resolution among the many components present in the oxidation reactions of the CAs. This was achieved mainly because the retention on the column of the CA starting molecules and the chromophores formed in the initial phase of the reactions was determined by adsorption phenomena in addition to the size exclusion phenomena. The combination of adsorption and size exclusion increased the resolution but it also increased the analysis time. SEC is not routinely applied to or recommended for the analysis of MNs.[6] However, in a previous report we described the application of SEC in the analysis of the MN derived from homogentisic acid[8]. As for the case of the CAs, the homogentisic acid starting compound exhibited additional retention phenomena on the SEC column. About twenty years ago, SEC was applied to the formation of MN from homogentisic acid by David et al.[9], using the same type of column and observing the same resolution pattern as in our previous report.[8] However, the application of SEC for the study of the MN formation appeared not to have been explored further. In addition to CAs and homogentisic acid, we have applied SEC in the study of the synthesis of MN from DOPA, catechol, pyrogallol, caffeic acid, chlorogenic acid, serotonin and others and these observations will be reported upon elsewhere. Thus, we would like to suggest the SEC analysis as a valuable approach to study the synthesis of MNs. As illustrated in Figure 6, select PS appeared to promote the auto-oxidation of CAs into MN; even in the absence of Cu2+. SEC analyses of reaction mixtures involving PS (see Figures 4 and 7) revealed that two fractions of high molecular mass materials are generated. One fraction has a retention time in region I of the SEC profiles, while the second fraction has a retention time in region II, often at similar retention times as for the native PS material used for the reaction. Both
fractions exhibit absorbance in the visible region of the spectrum. The FT-IR spectra comparing the analysis of the native PS and the MN material generated (see Figure 8) reveal very little qualitative differences, suggesting that the final product generated consists mainly of PS material with a limited presence of MN material, despite the obvious physical differences between the materials (white powder vs darkly colored, fibrous material). Similar observations were made for any MN synthesized from NE, DA or EPI in the presence of any type of PS. All these results strongly suggest that in the presence of PS, PS/MN complexes and aggregates are formed during the reactions. Whether the MN is complexed to the PS through covalent or non-covalent interactions remains unclear at this point. SEC analysis did provide assurances that the final MN materials generated through reactions with PS were free from low molecular starting compounds or other reaction intermediates following the dialysis purification process. The study of MNs touches many clinical or pathological issues and the possible link between soluble or deposited MNs and various diseases has been reviewed.[10] MNs have been implicated in the pathophysiology of, e.g., alkaptonuria[11] or Parkinson’s disease[4]. In addition, the presence of MNs has been implicated in the pathogenesis of certain bacterial or fungal species.[12-14] SEC, particularly with more advanced detectors like multi angle laser light scattering detection, can provide a powerful tool to study the synthesis of MNs from select precursors or to study the inhibition of the synthesis of MNs. In addition to the evaluation of MNs, SEC could similarly be applied to the study of other polyphenolic pigments, e.g., tannins or lignins, as the biochemistry and physic-chemical properties of these pigments share similarities with the biochemistry and physic-chemical properties of MNs.[15, 16]
Conclusions We have demonstrated the versatility of size exclusion chromatography in the study of the synthesis of melanins from catecholamine starting compounds. In addition, we demonstrated that polysaccharides can promote the oxidation of catecholamines to melanins and form high molecular mass polysaccharide/melanin complexes.
Acknowledgements This research and Astiney Clark were in part supported by a grant from the US Department of Education [#P031B090214]. Majidah Alhumaidi was supported by the Saudi Arabian Cultural Mission to the USA.
References [1] P. Meredith, T. Sarna, The physical and chemical properties of eumelanin, Pigment Cell Res, 19 (2006) 572-594. [2] J.D. Simon, D.N. Peles, The Red and the Black, Accounts of Chemical Research, 43 (2010) 1452-1460. [3] F. Solano, Melanins: Skin Pigments and Much More—Types, Structural Models, Biological Functions, and Formation Routes, New Journal of Science, 2014 (2014) 28. [4] H. Fedorow, F. Tribl, G. Halliday, M. Gerlach, P. Riederer, K.L. Double, Neuromelanin in human dopamine neurons: comparison with peripheral melanins and relevance to Parkinson's disease, Prog Neurobiol, 75 (2005) 109-124. [5] K.G. Usunoff, D.E. Itzev, W.A. Ovtscharoff, E. Marani, Neuromelanin in the human brain: a review and atlas of pigmented cells in the substantia nigra, Arch Physiol Biochem, 110 (2002) 257-369. [6] M. d'Ischia, K. Wakamatsu, A. Napolitano, S. Briganti, J.C. Garcia-Borron, D. Kovacs, P. Meredith, A. Pezzella, M. Picardo, T. Sarna, J.D. Simon, S. Ito, Melanins and melanogenesis: methods, standards, protocols, Pigment Cell Melanoma Res, 26 (2013) 616-633. [7] S. Ito, K. Wakamatsu, Quantitative Analysis of Eumelanin and Pheomelanin in Humans, Mice, and Other Animals: a Comparative Review, Pigment Cell Research, 16 (2003) 523-531. [8] A.M. Taylor, K.P. Vercruysse, Analysis of Melanin-like Pigment Synthesized from Homogentisic Acid, with or without Tyrosine, and Its Implications in Alkaptonuria, JIMD Rep, DOI 10.1007/8904_2016_27(2016). [9] C. David, A. Daro, E. Szalai, T. Atarhouch, M. Mergeay, Formation of polymeric pigments in the presence of bacteria and comparison with chemical oxidative coupling—II. Catabolism of
tyrosine and hydroxyphenylacetic acid by Alcaligenes eutrophus CH34 and mutants, European Polymer Journal, 32 (1996) 669-679. [10] Z.L. Hegedus, The probable involvement of soluble and deposited melanins, their intermediates and the reactive oxygen side-products in human diseases and aging, Toxicology, 145 (2000) 85-101. [11] N.B. Roberts, S.A. Curtis, A.M. Milan, L.R. Ranganath, The Pigment in Alkaptonuria Relationship to Melanin and Other Coloured Substances: A Review of Metabolism, Composition and Chemical Analysis, JIMD Rep, 24 (2015) 51-66. [12] J.D. Nosanchuk, A. Casadevall, The contribution of melanin to microbial pathogenesis, Cellular Microbiology, 5 (2003) 203-223. [13] J.D. Nosanchuk, R.E. Stark, A. Casadevall, Fungal Melanin: What do We Know About Structure?, Frontiers in Microbiology, 6 (2015) 1-7. [14] S. Seyedmousavi, M.G. Netea, J.W. Mouton, W.J.G. Melchers, P.E. Verweij, G.S.d. Hoog, Black Yeasts and Their Filamentous Relatives: Principles of Pathogenesis and Host Defense, Clinical Microbiology Reviews, 27 (2014) 527-542. [15] G. Carletti, G. Nervo, L. Cattivelli, Flavonoids and Melanins: A Common Strategy across Two Kingdoms, Int. J. Biol. Sci., 10 (2014) 1159-1170. [16] J. Ralph, G. Brunow, P.J. Harris, R.A. Dixon, P.F. Schatz, W. Boerjan, Lignification: are Lignins Biosynthesized via simple Combinatorial Chemistry or via Proteinaceous Control and Template Replication?, Recent Advances in Polyphenol Research, Wiley-Blackwell 2009, pp. 36-66.
Legends to the figures
Figure 1: Raper-Mason scheme for the synthesis of eumelanin from DOPA or catecholamines. Figure 2: Visible region of the UV_Vis spectra of DA (solid line), EPI (dotted line) and NE (dashed line) after reaction with Cu2+. CAs (final concentration 1.0mM) were mixed with Cu2+ (final concentration 0.3mM) in water and kept at RT overnight. Figure 3: RP-HPLC profile (signal at 275nm) of reaction mixture involving EPI, Cu2+, with or without CS A. Reaction mixtures contained 0.25mM EPI, 0.03mM Cu2+ and no (dotted line) or 2.5mg/mL CS A (solid line) and were kept at RT for two weeks prior to dilution and analysis. Figure 4, panel A: SEC profiles (signal at 275nm) profile of reaction mixtures involving EPI, Cu2+, with or without CS C. Reaction mixtures contained 0.5mM EPI, 0.03mM Cu2+ and no (dotted line) or 2.5mg/mL CS C (solid line) and were kept at RT for one week prior to dilution and analysis. Figure 4, panel B: SEC profiles (signal at 400nm) profile of reaction mixtures involving EPI, Cu2+, with or without CS C. Reaction mixtures contained 0.5mM EPI, 0.03mM Cu2+ and no (dotted line) or 2.5mg/mL CS C (solid line) and were kept at RT for one week prior to dilution and analysis. Figure 5: AUC as determined by SEC of DA, DA-derived chromophore and CS C as a function of the reaction time. The reaction mixture contained 2.5mg/mL CS C, 1mM DA and 0.1mM Cu2+ and was kept at RT for up to one week. The plot shows the AUC of: (a) the peak corresponding to DA (squares; using signal at 275nm), (b) the peak corresponding to the
chromophore generated from DA (circles; using signal at 300nm) and (c) the peak corresponding to CS C (triangles; using signal at 275nm). Figure 6: Pigment solutions or suspension obtained from the reaction between DA and various substances. About 30mg DA was dissolved in 10mL water and about 100mg test substance (top, from left to right: none, CARRA, ALG; bottom, from left to right: CS A, CS C, FUCO) was added as a powder and the mixtures were left at RT for one week. Figure 7, panel A: SEC profile of dialyzed mixture involving the reaction between fucoidan and DA. The SEC profile shown at 230nm (solid line) and 400nm (dashed line) involves the reaction mixture discussed in Figure 6. The mixture was dialyzed extensively with water and analyzed following 10-fold dilution. Figure 7, panel B: UV_Vis spectra of the two mean peaks of the SEC profile shown in panel A. The solid line spectrum corresponds to the peak retention time of about 6.7 minutes and the dashed line spectrum corresponds to the peak retention time of about 8.9 minutes. Figure 8: Comparison of the FT-IR spectra of fucoidan and pigment generated through a reaction between fucoidan and DA. The dashed line represents the spectrum of fucoidan and the solid line represents the sample discussed in Figure 6 and Figure 7, panels A and B.
S1570-0232(17)30142-3 http://dx.doi.org/doi:10.1016/j.jchromb.2017.04.005 CHROMB 20540
To appear in:
Journal of Chromatography B
Received date: Revised date: Accepted date:
25-1-2017 22-3-2017 1-4-2017
Please cite this article as: Koen P.Vercruysse, Astiney M.Clark, Paola A.F.Bello, Majidah Alhumaidi, Using size exclusion chromatography to monitor the synthesis of melanins from catecholamines., Journal of Chromatography Bhttp://dx.doi.org/10.1016/j.jchromb.2017.04.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Using size exclusion chromatography to monitor the synthesis of melanins from catecholamines.
Koen P. Vercruysse*,§, Astiney M. Clark*, Paola A. F. Bello*, Majidah Alhumaidi* *
Department of Chemistry, Tennessee State University; 3500 John A. Merritt Blvd., Nashville,
TN 37209
§
Corresponding author: e-mail: [email protected]; phone: +1-615-963-5340
Highlights
SEC is a versatile technique to monitor synthesis of melanins.
Many polysaccharides promote oxidation of catecholamines.
Polysaccharides form high molecular mass aggregates with melanins.
Abstract We have employed size exclusion chromatography (SEC) to the study of the auto- and Cu2+mediated oxidation of the catecholamines, dopamine, epinephrine and norepinephrine, into melanins. We observed that, due to non-size exclusion-mediated effects, the catecholamines and some of the low molecular mass intermediates generated during the oxidation reactions, could be resolved from each other and from the high molecular mass pigment generated. Thus, SEC
allowed us to monitor the disappearance of the catecholamine starting compounds, the appearance and subsequent disappearance of the low molecular mass chromophores generated in the initial phase of the reactions and the appearance of the high molecular mass melanins. In the process of this research, we observed that many, mostly anionic polysaccharides (PS), enhanced both the auto- and Cu2+-mediated oxidation of all three catecholamines. SEC analyses of reaction mixtures involving PS suggested that very high molecular mass aggregates between PS and melanins can be generated. In addition, SEC analysis allowed us to verify the efficiency of the dialysis purification process employed to obtain pure and dried melanin materials for cellbiological studies.
1. Introduction The pigments generated in our experiments belong to the category of the melanins (MN) and related biomolecules. MN are ubiquitously found in nature and excellent reviews regarding their biosynthesis, chemistry, classification and functions have been written.[1-3] The MN found in animals are built from L-DOPA which is derived from L-tyrosine. These types of MN are broadly divided into eumelanins and pheomelanins. The pheomelanins differ from the eumelanins that the amino acid L-cysteine is built into its structure. The pheomelanins have not yet been the subject of our experiments. The biosynthesis of MN involves a sequence of oxidation, cyclization and polymerization reactions most commonly described by the RaperMason scheme and illustrated in Figure 1.[1] In a first phase of this reaction scheme, the catechol portion of the precursor is converted into the o-quinone oxidation product and such o-quinones often exhibit absorbance in the visible region of the electromagnetic spectrum. Catecholamines (CAs) like epinephrine (EPI), norepinephrine (NE) or dopamine (DA) are biomolecules derived
from L-DOPA and can undergo a similar sequence of reactions leading to neuromelanins or other melanins, similar as for L-DOPA leading to eumelanins (see Figure 1). CAs like DA or NE are considered to be the precursors of the neuromelanins found in select areas of the brain.[4, 5] MN types of pigments are also produced by plants, fungi and bacteria, but the precursors of some of these pigments are often nitrogen-free phenols like catechol, caffeic acid, homogentisic acid or others.[3] Given the fact that MN constitute a broad range of biomolecules derived from a broad range of potential precursors, the qualitative and quantitative analysis and characterization is challenging, primarily due to the fact that the naturally-occurring MN are often insoluble.[6] Chromatographic approaches to the analysis of MN often involve the degradation of the pigment and the subsequent analysis of the degradation products.[6, 7] In this report we demonstrate the versatility of size exclusion chromatography (SEC) for the study of the formation of synthetic MN from CA precursors. In addition, we applied SEC to study the synthesis of MN from CAs in the presence of polysaccharides (PS). We observed that many PS promoted the oxidation reaction and that water-soluble, high molecular mass pigment/PS are generated in the process. Finally, we employed SEC to verify the dialysis purification process of the MN generated in our experiments.
2. Materials and Methods 2.1 Materials Chondroitin sulfate type A (sodium salt from bovine trachea; 70% with counterbalance chondroitin sulfate type C), chondroitin sulfate type C (sodium salt from shark cartilage; 90% with counterbalance chondroitin sulfate type A), alginic acid sodium salt (Algin®, sodium
alginate), -carrageenan (commercial grade, type II), carboxymethylcellulose (low viscosity grade; sodium salt), fucoidan (from Fucus vesiculosus, dopamine.HCl, epinephrine.HCl and norepinephrine.HCl, CuCl2.2H2O were obtained from Fisher Scientific (Suwanee, GA). All other reagents were of analytical grade. 2.3 UV/Vis spectroscopy UV/Vis spectra of DA, NE and EPI were obtained using a DU 800 spectrophotometer from Beckman Coulter (Fullerton, CA) against water as the blank. 2.4 RP-HPLC analyses RP-HPLC analyses were performed on a UFLC chromatography system equipped with dual LC6AD solvent delivery pumps and SPD-M20A diode array detector from Shimadzu, USA (Columbia, MD). Analyses were performed on a BDS Hypersil C8 column (125X4.6 mm) obtained from Fisher Scientific (Suwanee, GA). Analyses were performed in isocratic fashion using a mixture of water:methanol:acetic acid (90:10:0.05% v/v) as solvent. The sample volume was 20L. Samples were diluted to a CA concentration of approximately 0.1mM. 2.5 Size exclusion chromatography (SEC) SEC analyses were performed on a Breeze 2 HPLC system equipped with two 1500 series HPLC pumps and a model 2998 Photodiode array detector from Waters, Co (Milford, MA). Analyses were performed using an Ultrahydrogel 500 column (300 X 7.8 mm) obtained from Waters, Co (Milford, MA) in isocratic fashion using a mixture of 25mM Na acetate:methanol:acetic acid (90:10:0.05% v/v) as solvent. Samples were diluted with SEC solvent, centrifuged and 20 L was injected. Samples were diluted as indicated in the text or figures.
2.6 Dialysis and freeze drying Select samples were dialyzed using Spectrum Spectra/Por RC dialysis membranes with molecular-weight-cut-off of 3.5kDa obtained from Fisher Scientific (Suwanee, GA). Select dialyzed materials were frozen overnight and dried using a Labconco FreeZone Plus 4.5L benchtop freeze-dry system obtained from Fisher Scientific (Suwanee, GA). 2.7 FT-IR spectral analysis FT-IR spectroscopic scans were made using the NicoletiS10 instrument equipped with the SmartiTR Basic accessory from ThermoScientific (Waltham, MA). Scans were taken with a resolution of 4 cm-1 between 650 and 4,000 cm-1 at room temperature using a KBr beam splitter and DTGS KBr detector. Each spectrum represents the accumulation of 24 scans.
3. Results 3.1. Preliminary observations When Cu2+ was added to aqueous solutions containing EPI, DA or NE, a change in color, from colorless to pink, red or orange, appeared within hours of mixing depending on the concentrations of Cu2+ or CA involved. Figure 2 illustrates the UV_Vis spectra of the chromophores thus generated. For all three compounds the UV_Vis spectrum exhibited a distinct absorbance maximum between 450 and 500nm. It was presumed that these chromophores generated in the initial phase of the reaction corresponded to the o-quinone oxidation products of the CA precursors. When select PS were added to such reaction mixtures then within hours, e.g., fucoidan, or after overnight reaction, e.g., chondroitin sulfate (CS) A or C, the colors of the mixtures darkened to yellow-brown (EPI and NE) or grey-black (DA) (results not shown). These
observations prompted us to speculate that the chromophore generated initially reacted further, generating other types of colored substances. The reaction of CAs in the presence of Cu2+, with or without PS, was evaluated using RP-HPLC analyses. Figure 3 shows a typical RP-HPLC profile of EPI after reaction with Cu2+ in the presence or absence of CS A. EPI had a retention time of about 6 minutes and following reaction with Cu2+ new peaks emerged with retention times between 1 and 2.5 minutes that were poorly resolved. One peak within the 1 to 2.5 minute cluster had a UV_Vis spectrum similar to the ones shown in Figure 2 and this peak was presumed to be the chromophore generated in the initial phase of the reaction. RP-HPLC analyses of similar reactions that included select PS showed additional peaks, with retention times around 1 minute, as shown in Figure 3. RP-HPLC did not provide any resolution between the PS and the various reaction products generated from the CAs. However, when performing reactions in the presence of PS, the peak in the RP-HPLC profile associated with the chromophore declined or disappeared when the reactions were monitored over longer reaction times at RT (results not shown). In addition, the pattern of peaks observed in the 1 to 2.5 minute cluster changed significantly when monitored over longer periods of time (one day or longer). As before, it was speculated that the chromophore generated in the initial phase, reacted further, generating other types of colored substances. For the study of the oxidation of EPI, NE or DA in the presence of PS, RP-HPLC can be used to monitor the disappearance of the starting molecule under various reaction conditions, but can not adequately be used to evaluate the appearance of the various reaction products. 3.2. SEC studies The oxidation reactions of CAs and subsequent formation of pigmented materials was extensively evaluated using SEC. Reaction mixtures containing 0.5mM EPI, 0.03mM Cu2+ and 0
or 2.5 mg/L CS C were kept at RT for one week and, following two-fold dilution with SEC solvent, analyzed. Figure 4, panels A (signal at 275nm) and B (signal at 400nm), compares the SEC profiles of these reactions. For discussion purposes we subdivided the SEC profiles into four different regions. Region I includes peak retention times between 5 and 7 minutes; peak retention times thought to be close to the upper exclusion limit of the column. Region II includes peak retention times between 7 and 13 minutes; peak retention times typically observed following the injection of PS solutions and associated with the size distribution profiles of the PS. Region III includes peak retention times between 13 and 15 minutes; peak retention times close to the lower exclusion limit of the column as determined through the injection of water or other small molecules. Region IV includes peak retention times above 15 minutes; peak retention times typical for molecules with retentions determined by adsorption phenomena in addition to size-exclusion phenomena. In Figure 4, panel A, the peak with retention time of about 20 minutes corresponds to EPI. We observed that the SEC peak retention times of all three CAs fell in region IV of the SEC profiles (typically between 18 and 22 minutes). In Figure 4, panel A, the peak with retention time of about 16 minutes exhibited an UV_Vis profile similar to the one shown in Figure 2 (dotted line). It was presumed that this peak corresponds to the chromophore generated in the initial phase of the reaction. The SEC profiles of the reaction mixtures for all three CAs showed peaks with retention times between 15 and 17 minutes with UV_Vis profiles similar to the ones shown in Figure 2. When EPI or any of the other CAs reacted with Cu2+ in the absence of any PS, a cluster of up to three resolved peaks were observed in region III of the SEC profiles (see Figure 4, panel A). These peaks did exhibit absorbance in the visible region of the spectrum as shown in Figure 4, panel B. No peaks were observed in regions I or II of the SEC profiles. In the presence of PS, in addition to the peaks in regions III and IV of the SEC profiles,
broad peaks were observed in region II and, depending on the type of PS and CA involved, sharp peaks were sometimes observed in region I of the SEC profiles. The peaks in regions I or II of these SEC profiles exhibited absorbance in the visible region of the spectrum as shown in Figure 4, panel B. For a reaction involving 2.5 mg/mL CS C, 0.1mM Cu2+ and 1mM DA, SEC analyses were performed on diluted aliquots from the reaction mixture taken at various time intervals after the start of the reaction. Figure 5 shows the area-under-the-curve (AUC) of: (a) the peak corresponding to DA (retention time about 20min; using signal at 275nm), (b) the peak presumed to be corresponding to the o-quinone chromophore generated from DA (retention time about 18min; using signal at 300nm) and (c) the peak corresponding to CS C (retention time about 11.5min; using signal at 275nm). Figure 5 shows a rapid decline in the AUC corresponding to DA in the initial phase of the reaction, followed by a much slower decline. The AUC corresponding to the chromophore generated from DA increased in the initial phase of the reaction and then declined significantly. The AUC corresponding to CS C steadily increased as a function of the reaction time. Large scale reactions were set up that enabled us to isolate, purify and characterize as much as possible the pigmented materials generated through the reaction between select PS and CA without the addition of Cu2+. Figure 6 provides a picture of one such experiment. In a well of a plastic cell culture dish, about 30mg DA was dissolved in 10mL water. About 100mg test substance, carrageenan, alginate, CS A, CS C or fucoidan, was added as a powder and the mixtures were left at RT. Within minutes (carrageenan or fucoidan) or hours (alginate, CS A or CS C) dark colors appeared in these mixtures. The mixtures were left at RT for one week. At that point the picture shown in Figure 6 was taken and the pigments were siphoned off for dialysis
and freeze drying. Figure 7, panel A presents the SEC profile, shown at 230nm and 400nm, of the dialyzed reaction mixture involving DA and fucoidan as discussed for Figure 6. Two main peaks can be observed, one with a retention time of about 6.7 minutes and one with a retention time of about 8.9 minutes. When fucoidan is analyzed by itself, the SEC profile exhibits a single peak with a retention time of about 9 minutes (results not shown). Figure 7, panel B, presents the UV_Vis spectra of both peaks. Both peaks exhibit a similar pattern: strong absorbance in the UV region with an exponential decline in absorbance in the visible region. No distinct absorbance maxima could be observed. When fucoidan is analyzed, its UV_Vis spectrum displays absorbance in the UV region and none in the visible region (results not shown). Upon freeze drying, the material obtained from the reaction between fucoidan and DA consisted of black fibrous material and Figure 8 compares the FT-IR spectra of fucoidan and this dried material.
Discussion Our visual observations and UV_Vis, RP-HPLC or SEC analyses discussed for figures 2 through 5 regarding the oxidation of CAs and the corresponding changes in color are in line with the Raper-Mason scheme (see Figure 1) for the oxidation of DOPA to low molecular mass intermediates and their subsequent conversion into high molecular mass pigment. UV_Vis spectroscopy can be used to monitor the formation of colored substances in the oxidation reactions of the CAs, but does not allow one to make a clear distinction between the low molecular mass chromophores and the high molecular mass MNs present in the reaction mixtures. As shown in this report, RP-HPLC can be used to monitor the presence and decline of the CA starting compound in the oxidation reactions as the peaks corresponding to these molecules are well resolved from the other peaks in the chromatogram. However, the peaks
corresponding to the low molecular mass chromophores and other reaction products, including the high molecular mass MNs, are not resolved as they exhibit very low retention times. SEC provided sufficient resolution among the many components present in the oxidation reactions of the CAs. This was achieved mainly because the retention on the column of the CA starting molecules and the chromophores formed in the initial phase of the reactions was determined by adsorption phenomena in addition to the size exclusion phenomena. The combination of adsorption and size exclusion increased the resolution but it also increased the analysis time. SEC is not routinely applied to or recommended for the analysis of MNs.[6] However, in a previous report we described the application of SEC in the analysis of the MN derived from homogentisic acid[8]. As for the case of the CAs, the homogentisic acid starting compound exhibited additional retention phenomena on the SEC column. About twenty years ago, SEC was applied to the formation of MN from homogentisic acid by David et al.[9], using the same type of column and observing the same resolution pattern as in our previous report.[8] However, the application of SEC for the study of the MN formation appeared not to have been explored further. In addition to CAs and homogentisic acid, we have applied SEC in the study of the synthesis of MN from DOPA, catechol, pyrogallol, caffeic acid, chlorogenic acid, serotonin and others and these observations will be reported upon elsewhere. Thus, we would like to suggest the SEC analysis as a valuable approach to study the synthesis of MNs. As illustrated in Figure 6, select PS appeared to promote the auto-oxidation of CAs into MN; even in the absence of Cu2+. SEC analyses of reaction mixtures involving PS (see Figures 4 and 7) revealed that two fractions of high molecular mass materials are generated. One fraction has a retention time in region I of the SEC profiles, while the second fraction has a retention time in region II, often at similar retention times as for the native PS material used for the reaction. Both
fractions exhibit absorbance in the visible region of the spectrum. The FT-IR spectra comparing the analysis of the native PS and the MN material generated (see Figure 8) reveal very little qualitative differences, suggesting that the final product generated consists mainly of PS material with a limited presence of MN material, despite the obvious physical differences between the materials (white powder vs darkly colored, fibrous material). Similar observations were made for any MN synthesized from NE, DA or EPI in the presence of any type of PS. All these results strongly suggest that in the presence of PS, PS/MN complexes and aggregates are formed during the reactions. Whether the MN is complexed to the PS through covalent or non-covalent interactions remains unclear at this point. SEC analysis did provide assurances that the final MN materials generated through reactions with PS were free from low molecular starting compounds or other reaction intermediates following the dialysis purification process. The study of MNs touches many clinical or pathological issues and the possible link between soluble or deposited MNs and various diseases has been reviewed.[10] MNs have been implicated in the pathophysiology of, e.g., alkaptonuria[11] or Parkinson’s disease[4]. In addition, the presence of MNs has been implicated in the pathogenesis of certain bacterial or fungal species.[12-14] SEC, particularly with more advanced detectors like multi angle laser light scattering detection, can provide a powerful tool to study the synthesis of MNs from select precursors or to study the inhibition of the synthesis of MNs. In addition to the evaluation of MNs, SEC could similarly be applied to the study of other polyphenolic pigments, e.g., tannins or lignins, as the biochemistry and physic-chemical properties of these pigments share similarities with the biochemistry and physic-chemical properties of MNs.[15, 16]
Conclusions We have demonstrated the versatility of size exclusion chromatography in the study of the synthesis of melanins from catecholamine starting compounds. In addition, we demonstrated that polysaccharides can promote the oxidation of catecholamines to melanins and form high molecular mass polysaccharide/melanin complexes.
Acknowledgements This research and Astiney Clark were in part supported by a grant from the US Department of Education [#P031B090214]. Majidah Alhumaidi was supported by the Saudi Arabian Cultural Mission to the USA.
References [1] P. Meredith, T. Sarna, The physical and chemical properties of eumelanin, Pigment Cell Res, 19 (2006) 572-594. [2] J.D. Simon, D.N. Peles, The Red and the Black, Accounts of Chemical Research, 43 (2010) 1452-1460. [3] F. Solano, Melanins: Skin Pigments and Much More—Types, Structural Models, Biological Functions, and Formation Routes, New Journal of Science, 2014 (2014) 28. [4] H. Fedorow, F. Tribl, G. Halliday, M. Gerlach, P. Riederer, K.L. Double, Neuromelanin in human dopamine neurons: comparison with peripheral melanins and relevance to Parkinson's disease, Prog Neurobiol, 75 (2005) 109-124. [5] K.G. Usunoff, D.E. Itzev, W.A. Ovtscharoff, E. Marani, Neuromelanin in the human brain: a review and atlas of pigmented cells in the substantia nigra, Arch Physiol Biochem, 110 (2002) 257-369. [6] M. d'Ischia, K. Wakamatsu, A. Napolitano, S. Briganti, J.C. Garcia-Borron, D. Kovacs, P. Meredith, A. Pezzella, M. Picardo, T. Sarna, J.D. Simon, S. Ito, Melanins and melanogenesis: methods, standards, protocols, Pigment Cell Melanoma Res, 26 (2013) 616-633. [7] S. Ito, K. Wakamatsu, Quantitative Analysis of Eumelanin and Pheomelanin in Humans, Mice, and Other Animals: a Comparative Review, Pigment Cell Research, 16 (2003) 523-531. [8] A.M. Taylor, K.P. Vercruysse, Analysis of Melanin-like Pigment Synthesized from Homogentisic Acid, with or without Tyrosine, and Its Implications in Alkaptonuria, JIMD Rep, DOI 10.1007/8904_2016_27(2016). [9] C. David, A. Daro, E. Szalai, T. Atarhouch, M. Mergeay, Formation of polymeric pigments in the presence of bacteria and comparison with chemical oxidative coupling—II. Catabolism of
tyrosine and hydroxyphenylacetic acid by Alcaligenes eutrophus CH34 and mutants, European Polymer Journal, 32 (1996) 669-679. [10] Z.L. Hegedus, The probable involvement of soluble and deposited melanins, their intermediates and the reactive oxygen side-products in human diseases and aging, Toxicology, 145 (2000) 85-101. [11] N.B. Roberts, S.A. Curtis, A.M. Milan, L.R. Ranganath, The Pigment in Alkaptonuria Relationship to Melanin and Other Coloured Substances: A Review of Metabolism, Composition and Chemical Analysis, JIMD Rep, 24 (2015) 51-66. [12] J.D. Nosanchuk, A. Casadevall, The contribution of melanin to microbial pathogenesis, Cellular Microbiology, 5 (2003) 203-223. [13] J.D. Nosanchuk, R.E. Stark, A. Casadevall, Fungal Melanin: What do We Know About Structure?, Frontiers in Microbiology, 6 (2015) 1-7. [14] S. Seyedmousavi, M.G. Netea, J.W. Mouton, W.J.G. Melchers, P.E. Verweij, G.S.d. Hoog, Black Yeasts and Their Filamentous Relatives: Principles of Pathogenesis and Host Defense, Clinical Microbiology Reviews, 27 (2014) 527-542. [15] G. Carletti, G. Nervo, L. Cattivelli, Flavonoids and Melanins: A Common Strategy across Two Kingdoms, Int. J. Biol. Sci., 10 (2014) 1159-1170. [16] J. Ralph, G. Brunow, P.J. Harris, R.A. Dixon, P.F. Schatz, W. Boerjan, Lignification: are Lignins Biosynthesized via simple Combinatorial Chemistry or via Proteinaceous Control and Template Replication?, Recent Advances in Polyphenol Research, Wiley-Blackwell 2009, pp. 36-66.
Legends to the figures
Figure 1: Raper-Mason scheme for the synthesis of eumelanin from DOPA or catecholamines. Figure 2: Visible region of the UV_Vis spectra of DA (solid line), EPI (dotted line) and NE (dashed line) after reaction with Cu2+. CAs (final concentration 1.0mM) were mixed with Cu2+ (final concentration 0.3mM) in water and kept at RT overnight. Figure 3: RP-HPLC profile (signal at 275nm) of reaction mixture involving EPI, Cu2+, with or without CS A. Reaction mixtures contained 0.25mM EPI, 0.03mM Cu2+ and no (dotted line) or 2.5mg/mL CS A (solid line) and were kept at RT for two weeks prior to dilution and analysis. Figure 4, panel A: SEC profiles (signal at 275nm) profile of reaction mixtures involving EPI, Cu2+, with or without CS C. Reaction mixtures contained 0.5mM EPI, 0.03mM Cu2+ and no (dotted line) or 2.5mg/mL CS C (solid line) and were kept at RT for one week prior to dilution and analysis. Figure 4, panel B: SEC profiles (signal at 400nm) profile of reaction mixtures involving EPI, Cu2+, with or without CS C. Reaction mixtures contained 0.5mM EPI, 0.03mM Cu2+ and no (dotted line) or 2.5mg/mL CS C (solid line) and were kept at RT for one week prior to dilution and analysis. Figure 5: AUC as determined by SEC of DA, DA-derived chromophore and CS C as a function of the reaction time. The reaction mixture contained 2.5mg/mL CS C, 1mM DA and 0.1mM Cu2+ and was kept at RT for up to one week. The plot shows the AUC of: (a) the peak corresponding to DA (squares; using signal at 275nm), (b) the peak corresponding to the
chromophore generated from DA (circles; using signal at 300nm) and (c) the peak corresponding to CS C (triangles; using signal at 275nm). Figure 6: Pigment solutions or suspension obtained from the reaction between DA and various substances. About 30mg DA was dissolved in 10mL water and about 100mg test substance (top, from left to right: none, CARRA, ALG; bottom, from left to right: CS A, CS C, FUCO) was added as a powder and the mixtures were left at RT for one week. Figure 7, panel A: SEC profile of dialyzed mixture involving the reaction between fucoidan and DA. The SEC profile shown at 230nm (solid line) and 400nm (dashed line) involves the reaction mixture discussed in Figure 6. The mixture was dialyzed extensively with water and analyzed following 10-fold dilution. Figure 7, panel B: UV_Vis spectra of the two mean peaks of the SEC profile shown in panel A. The solid line spectrum corresponds to the peak retention time of about 6.7 minutes and the dashed line spectrum corresponds to the peak retention time of about 8.9 minutes. Figure 8: Comparison of the FT-IR spectra of fucoidan and pigment generated through a reaction between fucoidan and DA. The dashed line represents the spectrum of fucoidan and the solid line represents the sample discussed in Figure 6 and Figure 7, panels A and B.