pubs.acs.org/Langmuir © 2009 American Chemical Society
Controlling Tyrosinase Activity on Charged Polyelectrolyte Surfaces: A QCM-D Analysis Michael V. Gormally, Rebecca K. McKibben, Malkiat S. Johal,* and Cynthia R. D. Selassie* Department of Chemistry, Pomona College, 645 North College Avenue, Claremont, California 91711 Received March 2, 2009. Revised Manuscript Received April 30, 2009 The quartz crystal microbalance (QCM) was used to monitor the immobilization of tyrosinase on polycationic and polyanionic precursor assemblies in situ and in real-time. The resulting enzymatic surfaces were then exposed to various flavonoids, and the degree of binding was measured using QCM. We show that enzyme activity is retained when immobilized on polycationic films (flavonoid binding observed), while the active site is blocked when assembled on a polyanionic film (no flavonoid binding to the enzyme). We rationalize these observations by considering a combination of interlayer interpenetration and strong electrostatic interactions between the polyelectrolyte and tyrosinase’s dicopper 2+center. Ion-pair formation between anionic moieties of the polyanion and the metal-coordinated active site is suggested as the dominant mechanism leading to the deactivation of tyrosinase. We are currently working to expand this research to achieve a more general theory of how various metal-coordinated enzymes react with polyelectrolyte surfaces of varying structural morphology, charge density, and chemical composition.
Introduction The action of tyrosinase-catalyzed flavonoid oxidation has become an important area of interest because of its prevalence and sometimes undesirable effects.1 The process causes browning in plants, leading to a drop in value and nutritional content. In humans, tyrosinase can be used in medicinal or cosmetic applications, but overabundance of the enzyme is also the cause behind cases of hyperpigmentation.2 Using UV spectroscopy, the kinetics of binding and oxidation between tyrosinase and various flavonoids have been well studied.3 Several methods of blocking tyrosinase’s active site through chelating of its copper center have been successful,4-6 and many compounds that competitively inhibit the enzyme’s oxidative activity have been identified.7 However, in the present study, we focus on inhibiting the catalytic process through manipulating the accessibility of the active site in situ. We present a model system in which the enzyme is immobilized on either a polycationic or polyanionic thin film and demonstrate the effect of charge on enzyme-substrate complexation as exemplified by the tyrosinase-flavonoid system. Immobilization techniques currently employed include chemisorption, Langmuir-Blodgett deposition, intermolecular crosslinking, adsorption, and entrapment. Recently, methods of layer-by-layer (LbL) electrostatic self-assembly (ESA)8-10 to *Authors to whom correspondence should be addressed. E-mail: malkiat.
[email protected] (M.S.J.);
[email protected] (C.R.D.S.). Fax: (909) 607-7726. Website: http://pages.pomona.edu/∼msj04747/. (1) Prota, G. Med. Res. Rev. 1988, 8, 525–556. (2) (a) Boss, P. K.; Gardner, R. C.; Janssen, B. J.; Ross, G. S. Plant Mol. Biol. 1995, 27(2), 429. (b) Constabel, C. P.; Bergey, D. R.; Ryan, C. A. Proc. Natl. Acad. Sci. U.S.A. 1995, 92(2), 407–411. (3) Rice-Evans, C. A.; Miller, N. J.; Paganga, G. Free Rad. Biol. Med. 1996, 20 (7), 933–956. (4) Hider, R. C.; Lerch, K. Biochem. J. 1989, 257(1), 289–290. (5) Behera, B. C.; Makhija, U. Curr. Sci. 2002, 82 (1), 61-66. (6) Park, Y. D.; Lyou, Y. J.; Hahn, H. S.; Hahn, M. J.; Yang, J. M. J. Biomol. Struct. Dyn. 2006, 24(2), 131–138. (7) Xie, L.; Chen, Q.; Huang, H.; Wang, H.; Zhang, R. J. Biochem. 2003, 68(4), 487–491. (8) Decher, G.; Hong, J. D.; Schmitt, J. Thin Solid Films 1992, 831, 210–211. (9) Decher, G. Science 1997, 277, 1232. (10) Lowack, K.; Helm, C. A. Macromolecules 1998, 31, 823.
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immobilize enzymes directly to the surface of polyelectrolyte multilayer (PEMU) films have gained attention.11 In the current study, assembly of these PEMUs are characterized using a quartz crystal microbalance with dissipation monitoring (QCM-D) which uses minute shifts in the frequency of an oscillating quartz crystal, on which the film is built, to determine mass changes by the Saurbrey approximation (eq 1),12 ΔF ¼ -
nΔm C
ð1Þ
where C = 17.7 ng/cm2 Hz for a 5 MHz quartz crystal and n represents the overtone number. The dissipation factor (D) of the film deposited onto the sensor surface is a reflection of its viscoelasticity. Dissipation is measured by the response of the crystal’s energy loss after the induced oscillation is stopped. In this work, QCM-D is used to observe the real-time immobilization of tyrosinase onto precursor polycationic and polyanionic films and the subsequent deposition of flavonoid on the enzyme surface. Tyrosinase catalyzes the hydroxylation of monophenols and the oxidation of o-diphenols forming o-quinones.13 Containing a dicopper 2+center, the structure of tyrosinase is found in three forms: met, oxy, and deoxy.14 Each of the three contains two active sites, and structures for each form have been proposed. In this work, we explore the specific interaction between the cationic active site of tyrosinase and underlying polyelectrolyte surfaces, and how this interaction affects the binding between the enzyme and flavonoid. As with certain other enzymes,15 it was found that tyrosinase binds to both polycationic and polyanionic precursor surfaces in an electrostatic self-assembly. We credit this property (11) Tripathy, S. K.; Jayant, K.; Singh, N. H.; MacDiarmin, A. G. Handbook of Polyelectrolytes and Their Applictions; American Scientific Publishers: Stevenson Ranch, CA, 2002; Vol. 1. (12) Sauerbrey, G. Z. Phys. 1959, 155, 206–222. (13) Van Gelder, C. W.; Flurkey, W. H.; Wichers, H. J. Phytochemistry 2007, 45 (7), 1309–23. (14) Sanchez-Ferrer, A.; Rodrigues-Lopez, J. N.; Garcia-Canovas, F.; GarciaCarmona, F. Biochim. Biophys. Acta 1995, 1247(1), 1–11. (15) Hamlin, R. E.; Dayton, T. L.; Johnson, L. E.; Johal, M. S. Langmuir 2007, 23(8), 4432–4437.
Published on Web 06/08/2009
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to the enzyme’s abundance of both local cationic and anionic sites from its amino acid moieties. It was additionally found that, by depositing the enzyme on a cationic precursor film of poly (ethylenimine) (PEI), tyrosinase’s activity was preserved and subsequent binding of the flavonoid substrate was successful. However, deposition of the enzyme on an anionic precursor film, poly[1-[4-(3-carboxy-4-hydroxyphenylazo)benzenesulfonamido]-1,2-ethanediyl, sodium salt] (PAZO), prevented enzymesubstrate formation. We demonstrate that the ability to control tyrosinase’s activity based on the charge of precursor polyelectrolyte film holds for several polymers and propose a model for cationic stabilization of enzyme activity.
Experimental Section
as indicated. The steps were considered to be complete when F and D stopped changing with time. The flow rate for each experiment was 150 μL/min and controlled by a peristaltic pump (Ismatec ISM935C). All experiments were conducted at a cell temperature of 18 ( 0.02 °C. UV-visible absorbance spectra were collected using a Varian Cary 300 spectrophotometer. Glass substrates were prepared by immersion in a 30:70 H2O2(30% weight)/H2SO4 mixture for 1 h at 80 °C (piranha etch treatment). This treatment exposes free silanol (-Si-OH) groups on the substrate surface, which subsequently deprotonate in higher pH solutions (pH>3), thus resulting in an overall negatively charged surface. Following this treatment, substrates were rinsed thoroughly in water, sonicated for 15 min to remove any remaining etch solution, and then stored in water. Prior to film deposition, substrates were rinsed thoroughly in water and dried under a stream of nitrogen gas. Films were built by sequentially exposing the substrate to the polyelectrolyte or enzyme. For each layer, the glass substrate was immersed in the solution for 10 min and rinsed for 2 min in PBS.
PEI (MW=25 000 g mol-1, CAS 9002-98-6, mixture of linear and branched), PAZO (average MW ≈ 65 000-100 000 g mol-1, CAS 24615-84-7), and poly(styrene) sulfonate (PSS) (average MW=1 000 000 g mol-1, CAS 25704-18-1) were obtained from Aldrich and aqueous solutions were prepared at 10 mM (using the molecular weight of the monomer unit) in phosphate buffered solution (PBS, pH 7, Fisher). The flavonoids catechin, epicatechin, taxifolin, and myricetin were also obtained from Aldrich and prepared at 1 mM (myricetin was prepared at 0.1 mM due to its lower solubility) in PBS. Mushroom tyrosinase was obtained from Worthington and prepared at a concentration of 5.6 10-4 mmol in PBS. See the Supporting Information for molecular structures of all polyelectrolytes and flavonoids used as well as a representation of tyrosinase’s active site and catalysis of a phenolic compound. Frequency and dissipation data of the deposition of flavonoids on PEI surfaces were obtained using a quartz crystal microbalance with dissipation (QCM-D) monitoring (E4, Q-Sense, Gothenburg, Sweden). The QCM-D sensor consisted of an ATcut piezoelectric quartz crystal disk, coated with a Au electrode (100 nm thick) on the back and an active surface layer of SiOz (∼50 nm thick). The QCM-D sensor crystal (14 mm 0.3 mm, active area of 0.2 cm2) operated a frequency of 4.95 MHz ( 50 Hz. The crystals were optically polished with a surface roughness of less than 3 nm (root-mean-square). The active side was in contact with the aqueous solutions of PEI and flavonoids in PBS. The crystal was mounted in a flow cell with a total volume of 40 μL. The QCM-D operates by inducing an oscillation in the crystal at its fundamental resonant frequency. As matter is deposited onto the sensor surface, the resonant frequency of the oscillating crystal is decreased. If the mass deposited on the oscillator is sufficiently small in comparison to the crystal itself, and if the material is rigidly fixed onto the surface, the QCM-D is able to act as an extremely sensitive microbalance. The change in resonant frequency (ΔF) can be used to calculate the change in mass at the surface (Δm), including water coupled to the film, using the Saurbrey relation (eq 1). The third overtone values were used for frequency and dissipation measurements. At this overtone, the frequency values were obtained with an accuracy of (0.2 Hz and the dissipation factor with accuracy of (0.210-6 or less. F and D values were measured relative to a baseline obtained in PBS. Details of QCM-D principles and operation can be found elsewhere.16 Before each run, the SiO2-coated quartz crystals were decontaminated by UV/ozone treatment for 10 min, soaking in 2% Hellmanex solution (Hellma Co.) for 30 min, followed by a second UV/ozone treatment for 10 min, with the final two steps followed by an ultrapure water rinse and drying with N2. Ultrapure water was also flushed through the QCM-D flow cells until a stable baseline was achieved. During the experiments, solutions of PEI and flavonoids in PBS were passed through the QCM-D flow cells
In considering an electrostatically driven deposition of the enzyme on a charged precursor PEMU, its net charge was taken into account. The isoelectric point (pI) of tyrosinase has been reported to lie at pH=6.1.17 In the PBS buffer solution (pH=7), in which all reactions took place is 7, the net charge of tyrosinase will be slightly negative. However, because of the numerous amino groups on proteins, there will exist local regions of both positive and negative charges on tyrosinase. The presence of these locally charged regions suggests enzyme deposition will proceed on both cationic and anionic surfaces. The binding of tyrosinase to a PEI or PAZO surface was measured by QCM which provides the resonant frequency change (deposited mass) as a function of time. Since flavonoids will bind specifically to the active site on the surface immobilized tyrosinase, QCM can be used to monitor the adsorption and formation of the enzyme-substrate complex in real time. Figure 1a shows the decrease in frequency corresponding to each binding event. The initial drop in frequency from ∼10 to ∼50 min (∼12 Hz) indicates the adsorption of PEI to the underlying SiO2 substrate. The shaded areas in Figure 1 represent solute free buffer rinses. The second drop in frequency (∼30 Hz) from ∼60 to ∼100 min is more gradual and due to the deposition of tyrosinase onto the PEI surface. We can correlate these negative frequency shifts to mass depositions based on the Sauerbrey model.12 Dissipation values, which are a function of the rigidity of the film, increase very slightly during the entire deposition of both species, allowing us to accurately apply the Sauerbrey model. Thus, the first abrupt frequency shift due to PEI adsorption corresponds to a mass deposition of ∼212 ng/cm2, while the more gradual shift corresponds to a net deposition of ∼530 ng/cm2. In cases for which dissipation measurements did not remain near zero, we used the Sauerbrey model qualitatively in order to correlate decreases in frequency with increases in mass, although a specific number estimation cannot be accurately determined. Figure 1b shows the frequency change due to the deposition of epicatechin on the enzyme surface (denoted here as PEI-Ty to emphasize the presence of the underlying polycation). Deposition of the flavonoid occurs within 10 min, giving a net frequency change of 4 Hz (or ∼71 ng/cm2), indicating that epicatechin binds specifically to tyrosinase. Flowing pure buffer over the epicatechin surface resulted in complete desorption of the
(16) Todahl, M.; Hook, F.; Krozer, A.; Brezezinski, P.; Kasemon, B. Rev. Sci. Instrum. 1995, 66, 3924–3930.
(17) Matoba, Y.; Kumagi, T.; Yamamoto, A.; Yoshitsu, H.; Sugiyama, M. J. Biol. Chem. 2006, 281, 8981–8990.
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Results and Discussion
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Figure 1. (a) Deposition of a cationic PEI monolayer and the immobilization of tyrosinase on that layer. (b) Subsequent exposure of the cationically immobilized tyrosinase to epicatechin, leading to flavonoid binding, measured by QCM-D. The absorption of mass to the sensor surface is approximately proportional to the reported ΔF. Buffer rinses are indicated in the gray sections. Frequency shifts in this region are due to loss of extra, unbound material on the sensor surface.
flavenoid over a period of ∼15 min (not shown), likely due to the release of oxidized product. Figure 2a shows the effect of exposing tyrosinase to the anionic PAZO surface. The PAZO film was deposited by ESA to an underlying polycationic (PEI) film. The adsorption of the enzyme is slower than adsorption onto PEI and results in a net mass of deposition of ∼885 ng/cm2. Despite deposition of tyrosinase on the polyanion, no subsequent epicatechin deposition was observed on the enzyme (Figure 2b). In fact, a slight increase in frequency (or mass decrease of ∼6 ng/cm2) was observed, likely due to the loss of some enzyme. Thus, we confirm that deposition of tyrosinase proceeds on both a polycationic and a polyanionic surface, due to the abundance of various local charges. Greater depositions of the enzyme were observed on the polyanion. The greater degree of branching in PAZO likely leads to a greater degree of interlayer mixing with the enzyme.18 Additionally, we expect a strong electrostatic interaction between the polymer’s anionic sites and the dicopper 2+center of the enzyme, (18) Lane, T. J.; Fletcher, W. R.; Gormally, M. V.; Johal, M. S. Langmuir 2008, 24(19), 10633–10636.
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Figure 2. (a) Deposition of an anionic [PEI/PAZO] bilayer and the immobilization of tyrosinase on that bilayer. (b) Subsequent exposure of the anionically immobilized tyrosinase to epicatechin, leading to no flavonoid binding, measured by QCM-D.
leading to greater amount of interlayer interpenetration between the two films. Figures 1b and 2b show that epicatechin is able to bind with tyrosinase immobilized on cationic but not anionic films. Control runs in Figure 3 indicate that the substrates do not deposit on bare polyelectrolyte layers void of enzyme, regardless of the precursor’s surface charge. This indicates that the substrate’s association with the enzyme layer is not a self-assembled electrostatic attraction, as is the case with polyelectrolyte bilayers, but rather a substrate complexation with an enzyme active site. To confirm that such an association with the active site exists, UV-visible spectra of flavonoid solutions after exposure to the enzymatic surface were collected. Using the same solution parameters as described above, glass slides were coated with polymer and enzyme by dipping in the corresponding solution for 10 min (with 2 min buffer rinse cycles). The assemblies were subsequently allowed to sit in an epicatechin solution for 72 h. Figure 4 shows the spectra of two tyrosinase-capped films, one on a monolayer of cationic PEI and a second on a bilayer capped with anionic PAZO. The graph shows the formation of oxidized product, denoted by absorbance at oxidized product λmax, previously determined to be 436 nm. Oxidized product formed in the solution exposed to the tyrosinase when it had been immobilized on PEI, but minimal change in absorbance at 436 nm was observed for enzyme on PAZO, even after 3 days. This establishes that the substrate does indeed access the active site of tyrosinase contained Langmuir 2009, 25(17), 10014–10019
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Figure 3. Exposure of (a) PEI and (b) PAZO layers to epicatechin without immobilized tyrosinase, showing that no flavonoid deposits on bare polymer, measured by QCM-D.
Figure 4. Absorbance spectra of the product of oxidation of epicatechin catalyzed by tyrosinase immobilized on PEI (black line) and PAZO (gray line) precursors. Spectra demonstrate that tyrosinase immobilized on PEI allows for enzyme substrate interaction and subsequent catalysis of epicatechin to its oxidized product, while PAZO largely blocks that interaction.
in a film capped by PEI, and shows that binding of the substrate indicated in QCM data constitutes formation of an enzyme substrate complex. It is also worth mentioning that in systems where the flavonoid was able to bind to the film surface, a direct comparison of Langmuir 2009, 25(17), 10014–10019
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molecules of deposited flavonoid to immobilized tyrosinase indicates a ratio = 50:1, flavonoid/tyrosinase. Since control runs in Figure 3 rule out the possibility of substrate-polymer binding, other processes must be responsible for the larger than expected deposition of flavonoid. It is important to realize that QCM does not discriminate between the solute and the amount of hydration in the film. Therefore, we suggest the possibility of water hydration leading to an overreporting of mass during the flavonoid deposition step, or an underreporting of mass during the deposition of the enzyme on the polyelectrolyte. For instance, the greater degree of hydrophobicity of the enzyme may cause the underlying polyelectrolyte layer to deswell. However, it is significant that substrate binding and subsequent product formation (as indicated in Figure 4) remain unique to systems in which the enzyme is deposited on a cationic precursor. Thus, any additional binding of substrate is less important, as the ability of the flavonoid to specifically bind to the active site has been established. Further considering the behavior of tyrosinase in its immobilized state, since different sites on the enzyme (presumably of opposite charge) must be involved for association with either a negative or a positive polymer layer, it follows that tyrosinase immobilizes in different orientations depending on the charge of the underlying polymer layer . Furthermore, the different orientations must result in favorable exposure of the active site for cases in which the enzyme was immobilized on PEI, and blockage in the case of PAZO. It is well established that the dinuclear copper center of the active site of tyrosinase is flexible during catalysis.19 Thus, we suggest two contributing factors to explain why an anionic immobilization does not allow for enzyme-substrate interaction: ion-pair formation and interlayer interpenetration. The two-atom Cu2+ active site of tyrosinase does not experience attractive interaction with a polycationic surface, while a polyanionic layer feels a strong electrostatic attraction to it and tends to incorporate the enzyme into the film. The interpenetration of multilayer assemblies has been well studied and extends to other charged macromolecular systems such as proteins.20-22 Such interpenetration would enable significant contact, and hence interlayer mixing, between relatively flexible charged units embedded within the enzyme and the polyelectrolyte. Polyanionic precursor films undergo more extensive interpenetration with enzyme containing the flexible Cu2+ sites, consequently blocking them beneath molecules of the precursor. It is likely that additional reasons such as steric interactions or hydrophobicity are also responsible for this behavior. However, the proposed model is consistent with the behavior seen in experimental data gathered in our laboratory as well as data from numerous previous studies of immobilization of different enzymes on polycations and polyanions. In further testing the aforementioned electrostatic model, we exposed epicatechin to a tyrosinase surface that had been assembled on a trilayer composed of PEI/PAZO/PEI (Figure 5). Deposition of the substrate was observed resulting in a net mass increase of ∼124 ng/cm2, indicating that tyrosinase immobilizes on a cationic trilayer precursor film in a fashion analogous to the enzyme on a PEI monolayer. Both of these immobilizations on a (19) Alikhan, M. A. Comp. Biochem. Physiol. 1976, 54B, 37–42. (20) Decher, G., Schlenoff, J. B., Eds. Multilayer Thin Films: Sequential Assembly of Nanocomposite Materials; Wiley-VCH: Weinheim, Germany, 2003. (21) Lvov, Y.; Decher, G.; Haas, H.; Mohwals, H.; Kalachev, A. Phys. B 1994, 198, 89. (22) Houssam, W. J.; Schlenoff, J. B. Macromolecules 2005, 38, 8473-8480; 9566.
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Figure 5. (a) Deposition of a cationic [PEI/PAZO]PEI trilayer and immobilization of tyrosinase on that trilayer. (b) Subsequent exposure of the cationically immobilized tyrosinase to epicatechin, leading to flavonoid binding, measured by QCM-D.
Figure 6. (a) Deposition of an anionic [PEI/PSS] bilayer and immobilization of tyrosinase on that bilayer. (b) Subsequent exposure of the anionically immobilized tyrosinase to epicatechin, leading to no flavonoid binding, measured by QCM-D.
cationic precursor film allow for subsequent substrate access to the active site, suggesting that only the terminal polymer layer is involved in immobilization. Additionally, we have demonstrated similar precursor charge effects on immobilized enzyme activity for a second polyanion, polystyrene sulfonate (PSS). Figure 6a shows how the deposition of tyrosinase on a [PEI/PSS] bilayer prevents subsequent binding of epicatechin (Figure 6b). The deactivation of tyrosinase on a polyanion, having been repeated for a second polyanion, further suggests that the determining factor for flavonoid binding is precursor surface charge and not other properties unique to any one polymer. We are currently working with several different polycationic and polyanionic compounds to expand this generalization. Thus far, all results have mimicked the behavior seen in the reported PEI/PAZO experiments. In addition, control runs in Figure 3 indicate that when flavonoid was exposed to negative and positive polymer precursors, void of any immobilized enzyme, no deposition of flavonoid resulted. These trials confirm that binding of flavonoid is specific to the active site on tyrosinase, does not occur on bare polymer layers, and proceeds only when tyrosinase is immobilized on a cationic precursor. We found that catalytic activity for cationically immobilized tyrosinase was preserved for all of the flavonoids included in
Table 1. Summary of QCM-D Data Reporting the Outcome of Exposing Various Enzymatic or Polyionic Surfaces to Each of the Four Substratesa
(23) Mckibben, R.; Johal, M.; Selassie, C. Unpublished results.
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catechin
epicatechin
taxifolin
myricetin
[PEI] [PEI/PAZO] 2 2 2 71 ng/cm 124 ng/cm 71 ng/cm2 [PEI]-Ty 90 ng/cm [PEI/PAZO]-Ty [PEI/PSS]-Ty [PEI/PAZO/PEI]-Ty 106 ng/cm2 124 ng/cm2 106 ng/cm2 90 ng/cm2 a
The mass of substrate binding to these surfaces is reported for all four flavonoids. A (-) indicates that the flavonoid did not deposit on the surface. These data demonstrate the ability of all four flavonoids to access tyrosinse’s active site when the enzyme is immobilized on a cationic precursor film.
this study. All of these flavonoids are known to undergo tyrosinase-catalyzed oxidation in solution.23 Table 1 reports the results of deposition of catechin, epicatechin, taxifolin, and myricetin for all enzymatic or bare polymer layers tested as well as mass deposition data for flavonoids binding to tyrosinase immobilized on a PEI precursor. By including these four flavonoids in the study, we demonstrate the ability of this immobilization to expose the active site to various types of molecules. For example, the flavonol myricetin is planar, has higher electron density on the B-ring, and is quite rigid due in part to the presence of a carbonyl bond on the C-ring. In contrast, catechin is a flavan-3-ol. Langmuir 2009, 25(17), 10014–10019
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Its structure is bent and has less electron density on the B-ring, which is trans to its adjacent -OH group. Epicatechin and taxifolin are also similar compounds with slight differences in structure, rigidity, and electron density. Since immobilization of tyrosinase on a cationic surface preserves its oxidative activity for several structurally different flavonoids, we claim that this method of enzyme deposition is particularly effective. Furthermore, as a consideration, it has been suggested elsewhere that certain enzyme immobilizations can lead to an increase and optimization of catalytic activity.24 We postulate that this observation will also be true for tyrosinase immobilized on PEI precursors. Drawing from our model of cationic precursor immobilization, we suggest a justification: Formation of an enzyme-substrate complex between tyrosinase and a flavonoid initiates an oxidation process.6 During the formation of the enzyme-substrate complex, electron density must be relieved from the B-ring of the flavonoid. In a polycationic immobilization, there is a large electronic potential difference between the positive precursor and the negative electron density of the B-ring. We believe that this potential aids the flow of electron density, thus stabilizing the enzyme-substrate complex and assisting product formation. We are currently conducting kinetic experiments to explore this theory.
Conclusion Using mono-, bi-, and trilayers of PEI and PAZO or PSS, we have shown that immobilization of tyrosinase proceeds on (24) Zhang, X.; Guan, R. F.; Wu, D. Q.; Chan, K. Y. J. Mol. Catal. B: Enzym. 2005, 33(1-2), 43–50.
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both polycationic and polyanionic precursor PEMUs. In addition, we have demonstrated that enzyme activity is retained in polycationic precursor immobilizations, while the active site is blocked in immobilizations onto polyanionic precursors. We presented a twofold explanation for this phenomenon that is consistent with and is supported by our findings. This explanation identifies decreased interpenetration and ion pair formation as the properties of polycationic immobilizations, which lead to a retention of enzyme activity. Currently, we are working to expand this research to achieve a more general theory of how various metal-coordinated enzymes react to immobilization on different polycationic and polyanionic surfaces. We hope to find further support for our conclusions of polycationic stabilization of enzyme activity. Acknowledgment. We would like to acknowledge support in part from NIH (1R15ES014812-CS). This work was also supported by the Pomona College Department of Chemistry. Note Added after ASAP Publication. This article was published ASAP on June 8, 2009. A correction has been made to equation 1. The correct version was published on June 15, 2009. Supporting Information Available: Molecular structures of all polyelectrolytes and flavonoids used as well as a representation of tyrosinase’s active site and catalysis of a phenolic compound. This material is available free of charge via the Internet at http://pubs.acs.org.
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