Langmuir 1998, 14, 1139-1147
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Enzymatic Tempering of a Mussel Adhesive Protein Film Douglas C. Hansen,*,† Sean G. Corcoran,‡,§ and J. Herbert Waite| EG&G Instruments, Inc., Oak Ridge, Tennessee 37831-2011, Code 6177, Naval Research Laboratory, Washington, D.C. 20375, and Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716 Received August 5, 1997. In Final Form: November 21, 1997 An adhesive protein isolated from the marine mussel Mytilus edulis has been adsorbed to highly oriented pyrolytic graphite and enzymatically oxidized to yield a sclerotized film that is tightly bound to the substrate. Atomic force microscopy was used to image the surface and the adsorbed biopolymer, indicating uniform coverage of the substrate within 1 s of adsorption time from solution. Film thicknesses of the adsorbed protein films were estimated to be 25 and 73 Å for the sclerotized films. Imaging forces ranging from e20 to g600 nN were used to determine the stability of adhesive protein films without and with added catechol oxidase, respectively. Results indicate that the resistance of adhesive protein films to displacement can be enhanced 10-fold by enzymatic oxidation. Oxidation presumably leads to formation of covalent crosslinks between adsorbed protein chains.
Introduction Many naturally occurring structural biomaterials that fascinate biomimeticists are sclerotized, or hardened, which are utilized by various organisms for specific applications. Among these are sporocysts, eggshells, egg capsules, insect cuticles, silks, shell matrix proteins, adhesives, and byssal threads.1 In at least some of these, sclerotization has been attributed to a maturational postsecretory process known as quinone tanning.2 Quinone tanning has four requisite ingredients: a polyphenol, an electron acceptor such as oxygen, a catalyst (often an oxidase), and cross-linkable macromolecules.3 Accordingly, the polyphenol is oxidized to a quinone, which “tans” or cross-links the macromolecules. Thus a chemical process initiates and induces a physical process, i.e., sclerotization. The trouble with studying sclerotization as a process is that while quinone formation and even protein cross-linking can be followed experimentally in vitro, these are at best a poor measure of the physical process. In the present study, we measure sclerotization in a mussel adhesive protein, Mytilus edulis foot protein (Mefp-1), directly. This protein hardens in the mussel byssus where it serves both adhesive and protective functions.4,5 The hardening may be caused by quinone tanning as all necessary ingredients for the process are present in the byssus: Mefp-1 (macromolecule) consists of many tandem repeats of a decapeptide sequence that contain 3,4-dihydroxyphenyl-L-alanine [DOPA] (polyphenol) which is acted upon by catechol oxidase (catalyst) in the presence of oxygen (electron acceptor). Alternatively, hardening could be caused by the multivalent intermolecular complexation of Fe(III) by peptidyl-DOPA groups * To whom correspondence should be addressed. † EG&G Instruments. ‡ Present address: Hysitron Inc., Nanomechanics Research Laboratory, 2010 E Hennepin Ave, Minneapolis, MN 55413. § Naval Research Laboratory. | University of Delaware. (1) Structure, Cellular Synthesis and Assembly of Biopolymers; Case, S. T., Ed.; Springer Verlag: Berlin, 1992. (2) Waite, J. H. Biol. Bull. 1992, 183, 178. (3) Waite, J. H. Comp. Biochem. Physiol. 1990, 97B, 19. (4) Waite, J. H. Int. J. Adhes. Adhes. 1987, 7, 9. (5) Benedict C. V.; Waite, J. H. J. Morphol. 1986, 189, 171.
in Mefp-1.6 As many as 50-75 of these catecholato-iron complexes can be formed with each molecule of Mefp-1 in vitro with log stability complexes as high as 42 at pH 7.0.7 By scrupulously avoiding metals, we can independently assess the effect of quinone tanning on the sclerotization of Mefp-1 in vitro. In a previous study, Filpula et al.8 observed that treatment of recombinant Mefp-1 with catechol oxidase in vitro led to aggregation or polymerization of the protein that was detectable by polyacrylamide gel electrophoresis. Here, the effect of catechol oxidase on adsorbed Mefp-1 was determined by making atomic force microscope (AFM) images of protein adsorbed onto freshly cleaved highly oriented pyrolytic graphite (HOPG) before and after treatment with the enzyme. This approach has the advantage of monitoring directly and dynamically the effect of enzyme treatment on the mechanical properties of Mefp-1. Experimental Section The images and measurements were made over various time intervals at room temperature using Digital Instruments III AFM in both Tapping and contact modes. Imaging forces were estimated from the force curves obtained with Digital Instruments software and using the supplied spring constants of the Digital Instruments cantilevers. Two different oxide-sharpened silicon nitride cantilevers of 100 and 200 µm lengths with spring constants of approximately 0.06 and 0.38 N/m, respectively, were used. Calibration and setting of the minimum applied repulsive forces in the Z plane were made prior to imaging.9 Estimated forces are intended to give relative differences in the applied loads only since calculating specific loads from AFM force curves is often difficult and unreliable. Isolation and purification of Mefp-1 followed the methods previously described.10 The amino acid composition of the purified protein was as follows in mol %: 1.6 Asp, 9.9 Thr, 7.9 Ser, 0.7 Glu, 6.1 Pro, 2.1 Gly, 7.0 Ala, 0.8 Val, 0.2 Met, 0.8Ile, 1.0 Leu, 17.4 DOPA, 4.7 Tyr, 0.1 Phe, 0.4 His, 18.8 Lys, and 0.4 (6) Taylor, S. W.; Luther, G. W., III; Waite, J. H. Inorg. Chem. 1994, 33, 5819. (7) Taylor, S. W.; Chase, D. B.; Emptage, M. H.; Nelson, M. J.; Waite, J. H. Inorg. Chem. 1996, 35, 7572. (8) Filpula, D. R.; Lee, S.-M.; Link, R. P.; Strausberg, S. L.; Strausberg, R. L. Biotechnology 1990, 6, 171. (9) Cullen, D. C.; Lowe, C. R. J. Colloid Interface Sci. 1994, 166, 102. (10) Rzepecki, L. M.; Nagafuchi, T.; Waite, J. H. Arch. Biochem. Biophys. 1991, 285, 17.
S0743-7463(97)00881-0 CCC: $15.00 © 1998 American Chemical Society Published on Web 01/30/1998
1140 Langmuir, Vol. 14, No. 5, 1998 Arg. Ten microliters of 0.05 M sodium phosphate (pH 7.0) was placed on a freshly cleaved sample of HOPG. Next, 3.5 µL of the purified mussel protein (1.0 mg/mL) in 5% (v/v) acetic acid was added and the solution was allowed to sit on the HOPG sample under a watchglass at room temperature for various time intervals. After the selected time period had elapsed, 10 µL of a 1.0 mg/mL solution of enzyme in distilled water was added to the buffer/protein solution; the surface area of the HOPG covered by the solution was 28 and 38 mm2 for protein/buffer and protein/ buffer/enzyme, respectively. Finally, after the elapse of another time interval, the buffer/protein/enzyme solution was removed by rinsing the sample with 2-3 mL of triple-distilled water from a wash bottle. Compressed ultrapure nitrogen gas was used to remove excess solution from the sample, which was then immediately placed on the AFM for imaging in contact or Tapping mode. The enzyme mushroom catechol oxidase (EC 1.14.18.1, also known as tyrosinase) was obtained from Sigma Chemical Company, St. Louis, MO, with a specific activity of 400 000 units/ mg. This enzyme was used instead of byssal catechol oxidase because of the latter enzyme’s insolubility and problematical purification.11,12 In any case, the two have similar catalytic activities toward o-diphenols including Mefp-1.13 Catechol oxidase (1.0 mg/mL) was thermally denatured by immersing a 1.5 mL centrifuge tube that contained a 1.0 mg/mL solution of enzyme into boiling water for 20 min. Enzyme inactivation was confirmed by monitoring the activity of the denatured enzyme on pyrocatechol (1.0 mg/mL at pH 7.0) for 20 min at room temperature as per Rzepecki and Waite.13
Results For clarity, all the images in Figure 1 were generated using the Tapping mode since the adsorbed protein was easily displaced by the AFM tip in the contact mode. A sample of HOPG rinsed with triple distilled water is presented in Figure 1A for comparison. As can be seen in Figure 1B, a 1 s adsorption time resulted in nearly uniform coverage of the HOPG by the mussel protein. Adsorption times of 15 s and 1 min (images C and D of Figure 1) resulted in more complete coverage with an approximate thickness layer of 29 Å after 1 min. This estimate was accomplished by imaging the surface in contact mode, which allowed the AFM tip on the 0.06 N/m cantilever to displace the adsorbed protein from the HOPG surface while scanning, and then using Tapping mode to compare the height of the undisturbed layer with that of the exposed HOPG (Figure 2). The thickness of the adsorbed protein did not appear to change as a function of adsorption time; a film thickness of 23 Å after a 5 min adsorption was not significantly different from that measured after 1 min (parts A and B of Figure 2). On the basis of these and other measurements (not shown), we concluded that a monolayer of Mefp-1 with an average approximate thickness of 25 Å was being deposited on the HOPG surface. This contrasts with the mean thickness of 55 Å measured by Olivieri et al.14 using surface ellipsometry on Cell-Tak, a commercial formulation in which Mefp-1 represents about 60 wt % of the protein. In their study, they measured an air-dried film at a surface concentration of 0.4 µg/cm2. In comparison, the surface concentration of the protein was 0.09 µg/cm2. In addition, comparison of the two images reveals an observable difference in the amount of displaced protein as a function of time with similar force loads estimated to be between 10 and 20 nN. This may be due to the auto-oxidation of the DOPA residues in the protein at neutral pH. This (11) Waite, J. H. J. Mar. Biol. Assoc. UK 1985, 65, 359. (12) Burzio, L. K. In Catechol oxidases associated with byssus formation in the blue mussel Mytilus edulis; Master’s Thesis, University of Delaware, Newark DE, 1996. (13) Rzepecki, L. M.; Waite, J. H. Anal. Biochem. 1989, 179, 375. (14) Olivieri, M. P.; Loomis, R. E.; Meyer, A. E.; Baier, R. E. J. Adhes. Sci. Technol. 1990, 4, 197.
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auto-oxidation anticipates the more rapid, catalyzed effect in the presence of catechol oxidase (Figure 2C). A 1 min adsorption of the protein followed by a 5 min tyrosinase treatment resembles the 5 min adsorption of Mefp-1 without enzyme (Figure 2B) in terms of protein displacement by the AFM tip with similar force loads (e20 nN). Treatment of adsorbed Mefp-1 with mushroom catechol oxidase for longer periods of time resulted in a marked change in both the morphology of the adsorbed protein film and its resistance to displacement by the AFM tip. We believe we have imaged the sclerotization process in action, whereby protein resistance to displacement increases with each scan over a given time period until there is no further displacement. This is presented in the series of images in Figure 3, where the protein has been adsorbed onto the HOPG for 1 min followed by treatment with the enzyme for 13 min and imaged using the 0.38 N/m cantilever in contact mode. In Figure 3A, the AFM tip has come into contact with the surface scanning in the upward direction beginning at the top half of the image area. The protein is displaced by the tip, thus revealing the underlying graphite (darker zones), with an atomic step of the graphite visible as a diagonal line across the image. As the AFM tip reaches the top of the scan and reverses direction (Figure 3B), it passes over the top half of the image already covered in the first scan and displaces more of the protein, resulting in an increase in visible graphite surface. After the fourth scan (Figure 3C) it can be seen that the protein in the top half has stabilized and no longer differs from that of the second scan (Figure 3B). In Figure 3D, the image area has been increased to show the original scanned area and surrounding undisturbed protein. This image was also generated in the contact mode, indicating that the surrounding area was not being displaced by the AFM tip. The elapsed time from addition of the enzyme to the completion of this image was approximately 22 min. Height comparison of the undisturbed protein versus the exposed HOPG indicates a vertical distance estimated at 73 Å. This is an increase in height compared to that of the adsorbed protein only, suggesting the enzyme may have adsorbed onto already adsorbed Mefp-1, resulting in a thicker film. Immediately following the scanning of the image in Figure 3D, the tip was moved to another area on the sample, and images were generated in contact mode (Figure 4A). Again, the protein was not displaced by the AFM tip, and focusing on a 2 × 2 µm area (Figure 4B) with the 0.38 N/m cantilever at maximum applied force on the order of several hundred nanometers (115-690 nN) resulted in minimal displacement of adsorbed Mefp-1. Continued scanning within a 10 × 10 µm area resulted in minimal disruption of the adsorbed protein (Figure 4C). On comparison of the estimated forces applied in parts A and B of Figure 2 with the estimated forces applied in Figure 4B, it is evident that adsorbed Mefp-1 resistance to displacement by the AFM tip increases when treated with catechol oxidase. This suggests a change such as sclerotization in the physical state of the protein. Adsorption of Mefp-1 onto HOPG for 1 min followed by a 20 min exposure to heat denatured catechol oxidase resulted in protein displacement at applied forces g20 nN (Figure 5A). In this case, height measurements indicate a film thickness of 40 Å, which is significantly lower than that of the active enzyme treatment. Adsorption of buffered Mefp-1 to HOPG for 20 min without enzyme (Figure 5B) required displacement forces similar to those measured using the heat-denatured enzyme. These results suggest that the denatured enzyme is simply adsorbing to the protein film and that auto-oxidation of
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Figure 1. Topography and phase AFM images of HOPG generated using Tapping mode: (A) freshly cleaved sample of HOPG rinsed with triple distilled water and dried under ultrapure nitrogen gas; (B) adsorption of Mefp-1 for 1 s onto HOPG from solution; (C) adsorption of Mefp-1 for 15 s; (D) adsorption of Mefp-1 for 1 min.
adsorbed Mefp-1 may be important in extended incubations and leads to an increased cohesiveness of the film.
Under identical buffer conditions and a 1 min Mefp-1 adsorption time followed by a 20 min catechol oxidase
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Figure 2. Topography and phase AFM images of Mefp-1 adsorbed onto HOPG from solution for 1 min (A), 5 min (B), and a 1 min adsorption followed by a 5 min treatment with mushroom tyrosinase (C). Images were generated by scanning in contact mode with the 0.06 N/m cantilever first, followed by imaging in Tapping mode. The center portion in each image is approximately 10 × 10 µm.
treatment yielded a thoroughly stabilized film. An estimated force load of 115 nN using a 0.06 N/m cantilever
in contact mode resulted in a compression of the scanned area. Analysis of the scanned area in contrast to the
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Figure 3. Topography AFM images generated in contact mode using a 0.38 N/m cantilever of Mefp-1 adsorbed onto HOPG for 1 min followed by a 13 min treatment with mushroom tyrosinase: (A) first scan where the AFM tip engages the surface; (B) second scan where the tip retraces the scanned area in (A) and continues scanning down into previously nonscanned material; (C) fourth scan over same area; (D) sixth scan of area enlarged to 13 µm showing previously scanned 10 × 10 µm area containing displaced protein inside of surrounding undisturbed cross-linked protein. Arrows denote direction of scan; rectangle indicates area of depth profile analysis (below).
adjacent undisturbed protein indicates that there is no difference in the height of the two, only a decrease in the
surface roughness of the scanned area (Figure 6). The ability of the adsorbed and catechol oxidase-treated
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Figure 4. Topography AFM images generated in contact mode using 0.38 N/m cantilever of the same sample in Figure 3 immediately following in another area: (A) second scan of a 10 × 10 µm area; (B) a fourth scan focusing in on a 2 × 2 µm area with maximum force being applied; (C) a sixth scan expanding out to a 10 × 10 µm scan area again showing the area scanned in (B) in the upper right corner.
biopolymer to undergo deformation such as compression by the AFM tip while remaining adsorbed to the HOPG surface suggests high levels of interfacial shear strength. Discussion These findings indicate that the addition of mushroom catechol oxidase to adsorbed Mefp-1 enhances resistance to displacement by force loads. In principle, this could be due either to the catalytic activity of the enzyme or to the coadsorption of enzyme with Mefp-1. Two controls militate against a coadsorption phenomenon: Mefp-1 resistance to displacement following treatment with heat-denatured enzyme resembles the nonenzymic control; moreover, even the nonenzymic controls tend to develop a resistance to displacement over time. More likely, the displacement resistance of Mefp-1 reflects the enzyme-catalyzed formation of extensive intermolecular cross-links between adsorbed Mefp-1 molecules. An increased thickening of the protein/enzyme film with the concomitant resistance to displacement would require the incorporation of the enzyme into the adsorbed protein film, or adsorption onto the protein film followed by cross-linking between enzyme and Mefp-1; otherwise shearing of the adsorbed enzyme
layer from the protein or separation of the protein/enzyme film from the HOPG surface would be observed. There is no doubt that peptidyl-DOPA residues of Mefp-1 are enzymatically oxidized to peptidyl quinones, but what follows in Mefp-1 remains unknown. Michael-type addition products between DOPA and lysine or histidine are well characterized in other proteins15 as have polyphenolic coupling reactions.16,17 Many of these cross-linked compounds, however, are not stable to complete hydrolysis especially when trace levels of transition metals, e.g., Fe are present. Given its in situ nondestructive capabilities, we expected solid-state NMR analyses of 13C and 15Nlabeled byssal threads to clinch the matter: Few if any nucleophilic adducts or phenolic coupling products were detected.18,19 These studies should have yielded the (15) Wang, S. F.; Mure, M.; Medzihradsky, K. F.; Burlingame, A. L.; Brown, D. E.; Dooley, D. M.; Smith, A. J.; Kagan, H. M.; Klinman, J. P. Science 1996, 273, 1078. (16) Anderson, S. O.; Jacobsen, J. P.; Bojesen, G.; Roepstorff, P. Biochim. Biophys. Acta 1992, 1118, 134. (17) Waite, J. H. Comp. Biochem. Physiol. B 1990, 97B, 19. (18) Holl, S. M.; Hansen, D.; Waite, J. H.; Schaefer, J. Arch. Biochem. Biophys. 1993, 302, 255.
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Figure 5. Topography AFM images generated in contact mode using 0.38 N/m cantilever of Mefp-1 adsorbed onto HOPG for 1 min followed by treatment with thermally denatured enzyme for 20 min and (B) Mefp-1 adsorbed onto HOPG for 20 min.
definitive conclusion regarding this matter were it not for the fact that the experimental designs for these studies may have been biologically flawed. Long incubation times of mussels in stationary seawater were used to achieve sufficient label uptake and incorporation into byssus prior to NMR. This may well have shielded the secreted byssal (19) Klug, C. A.; Burzio, L. O.; Waite, J. H.; Schaefer, J. Arch. Biochem. Biophys. 1996, 333, 221.
threads from the normal flow or turbulence required for oxidative chemical maturation.20 Our results with the AFM demonstrate that adsorbed Mefp-1 can be sclerotized or cohesively toughened by exposure to catechol oxidase. This enhances our understanding of the maturation of mussel adhesive proteins in vivo as well as contributing to the development of more (20) Dolmer, P.; Svane, I. Ophelia 1994, 40, 63.
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Figure 6. Surface plot of area treated with Mefp-1 and enzyme as described in text, showing the difference in surface roughness at the boundary of 10 × 10 µm area scanned in contact mode.
commercially relevant applications ranging from medical adhesives21,22 and metal scavenging agents6 to anticorrosive coatings.23,24 Although the toughening effect is (21) Burzio, V. A.; Silva, T.; Burzio, L. O. Anal. Biochem. 1996, 241, 190. (22) Schnurrer, J.; Lehr, C. M. Int. J. Pharm. 1996, 141, 251. (23) Hansen, D. C.; Dexter, S. C.; Waite, J. H. Corr. Sci. 1995, 37, 1423.
probably due to the enzyme-catalyzed formation of DOPA quinones followed by cross-link formation, the cross-links remain to be independently confirmed. If the toughening effect of catechol oxidase on adsorbed Mefp-1 had been limited to cohesive interactions alone, one might have expected the adsorbed film to delaminate at the higher (24) Hansen, D. C.; McCafferty, E. J. Electrochem. Soc. 1996, 143, 114.
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applied tip forces. The fact that this did not happen suggests that an enhancement of interfacial forces might be occurring as well. Acknowledgment. The authors gratefully acknowledge Dr. R. J. Colton of the Naval Research Laboratory for the use of the AFM and Dr. J. B. Green for his
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invaluable assistance in generating some of the AFM images. This work was performed while S.G.C. held a National Research Council-Naval Research Laboratory Research Associateship. J.H.W. acknowledges support from NIDR-Biomaterials. LA970881W
