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Articles Preparation of Insect-Cuticle-Like Biomimetic Materials Merle Miessner,† Martin G. Peter,*,† and Julian F. V. Vincent‡,§ Institut fu¨r Organische Chemie und Biochemie der Universita¨t Bonn, Gerhard-Domagk-Strasse 1, D-53115 Bonn, Germany; and Centre for Biomimetics, 1 Earley Gate, Reading RG6 6AT, U.K. Received December 21, 2000; Revised Manuscript Received February 21, 2001
A model system of tanning of a protein matrix within a fibrous structure, such as most commonly found in insect cuticle, was developed, using the cellulose of paper in place of chitin. The paper was impregnated with a tripeptide, DOPA-Gly-Gly, or a protein (BSA) plus catechol and treated with tyrosinase to oxidize the catechol. The resulting material was waterproof and had very high wet strength. If the material was wetted and dried repeatedly its water retention decreased by a factor of at least 2. Introduction There are many extracellular proteinaceous materials produced by invertebrate animals. Examples are insect cuticle, mussel byssus threads, periostracum of molluscs, and tubes and parts of tubes of numerous marine worms, coelenterates, and members of minor invertebrate phyla. In all these materials, mechanical stability and general chemical inertness are obtained by tanning the protein by the enzymatic oxidation of catechols (1,2-dihydroxybenzenes) of various types1-3 which are incorporated separately into the protein matrix. The catechols often have significant side chains attached which can affect their hydrophobicity. The basic chemistry of tanning by catechols has been summarized in many reviews.4-6 These catechols are commonly thought to work as direct cross-linking agents which stabilize the protein by holding the chains tightly together.1,2,7 However, the covalent crosslinking hypothesis does not account for all the effects of catechol tanning.8 In particular, it cannot account for the changes in the water content of the proteins.9,10 The catechols do not introduce sufficient cross-links to account for the increase in stiffness on tanning.8,11 The model cannot account for the resistance of tanned cuticle to rehydration and swelling.10 In addition, the oxidation of catechols such as N-acetyldopamine and N-β-alanyldopamine leads to their polymerization and in the presence of amino acids to the destruction of positive charges, in particular of the imidazole groups of histidine and of the -amino groups of lysine residues.12,13 Thus, the number of hydration sites is reduced * To whom all correspondence should be addressed. New address: Institut fu¨r Organische Chemie und Strukturanalytik der Universita¨t Potsdam, Karl-Liebknecht-Str. 25, D-14476 Potsdam, Germany. E-mail: peter@ serv.chem.uni-potsdam.de. † Institut fu ¨ r Organische Chemie und Biochemie der Universita¨t Bonn. ‡ Centre for Biomimetics. § New address: Department of Mechanical Engineering, The University, Bath, BA2 7AY, U.K.
in a tanned protein, and the addition of aromatic nuclei will further increase the hydrophobicity of the protein. There is no evidence for interchain (as opposed to intrachain) crosslinking of proteins although coupling of histidine residues of proteins to catechol has been demonstrated by NMR spectroscopy,14 and this possibility cannot be excluded with certainty. However, it is not crucial from the mechanical point of view.11 Most if not all of the biochemistry so far done on tanning systems has been in dilute solution. A more realistic model would have the components at much higher concentrations, perhaps on a solid substrate. It would also clearly be an advantage to have a model system which could be manipulated more easily than the simplest model which we have to date (isolated maggot cuticle). The experiments reported here were initiated to explore the possibility of using paper as a framework for depositing protein and tanning it, developing it into a model system for catechol tanning in general. Materials and Methods All substrates were dissolved in 500 mM potassium phosphate buffer pH 7.00 (standard buffer from BDH Laboratory Supplies). In particular, the following substances were used. The tripeptide DOPA-Gly-Gly (DGG) was synthesized by conventional methods and used at a concentration of 0.33 M. Bovine serum albumin and gelatin (100 mg mL-1) and polylysine (5 mg mL-1) were from Sigma. Casein (50 mg mL-1) was from MMB. The tanning agents phenol, catechol, 4-methylcatechol, tyrosine, and DOPA were from Sigma or from Aldrich. N-acetyldopamine was prepared as described by Koeppe and Mills.15 Tyrosinase (0.25 mg mL-1) was from Sigma. The tyrosinase inhibitor used was tropolone16 (Sigma) at 10 mM. The peptide coupling reagent used with DGG was N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) (Sigma). Nitrogen was determined using the Dumas method in a LECO FP228 protein analyzer.
10.1021/bm005652u CCC: $20.00 © 2001 American Chemical Society Published on Web 03/17/2001
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Figure 1. Effect of tanning by means of the tripeptide DGG or protein (BSA) on the water uptake of filter paper (Macherey-Nagel). Symbols: (C) control; (Tse) tyrosinase; (DGG) DOPA-Gly-Gly; (EDC) N-(3dimethylaminopropyl)-N′-ethylcarbodiimide; (BSA) bovine serum albumin; (Cat) catechol; (Me-Cat) 4-methylcatechol; (Y) L-tyrosine; (DOPA) 3,4-dihydroxyphenylalanine. The order in which the reagents were added is given in the text. Tyrosinase was always last.
Figure 3. Water retention of chromatography paper (Whatman) treated with gelatin, showing reduction in retention of water over a series of wetting (2 h in distilled water) and drying cycles. Filled circles: untanned. Filled squares: tanned with 2% phenol. Open squares: tanned with 2% catechol. The bars indicate (SD (five replicates).
Figure 2. Water retention of chromatography paper (Whatman) treated with BSA, showing reduction in retention of water over a series of wetting (2 h in distilled water) and drying cycles. Filled circles: 15 µg BSA, tanned with 2% catechol. Filled squares: 20 µg BSA, tanned with 2% catechol. Open squares: 20 µg BSA, tanned with 0.5% catechol. The bars indicate (SD (five replicates).
The filter paper used in the first series of experiments (results shown in Figure 1) was from Macherey-Nagel (Du¨ren, Germany), weighing 83 g m-2. In a second series of experiments (results shown in Figures 2-4) Whatman No. 4 chromatography paper weighing 92 g m-2 was used. The untreated filter paper strips measuring 13 × 100 mm were weighed, then wetted with 200 µL of a protein solution, and lightly blotted between two paper tissues to remove surface moisture. The moist paper strips were weighed to measure the volume of liquid absorbed and allowed to dry. During the oxidation and polymerization reactions, the test paper strips were placed between glass plates in order to avoid them drying out too quickly and allow the reaction to
Figure 4. Water retention of chromatography paper (Whatman) treated with BSA, showing reduction in retention of water over a series of wetting and drying cycles of different times of wetting. The bars indicate (SD (five replicates).
proceed. They were then washed for 1 min in distilled water and dried for 6 h. The dry paper strips were again weighed to measure the amount of absorbed material. The test strips were again moistened with 200 µL of water, blotted between paper tissues, and again weighed to determined the volume
Insect-Cuticle-Like Biomimetic Materials
of liquid absorbed. In the second series of experiments the paper was wetted by holding it in distilled water for 1 min. This wetting, weighing and drying was repeated a number of times according to the design of the experiment. The weight of water is expressed in g‚g-1 dry weight of the treated paper. Simple tensile testing was performed using an Instron tabletop universal testing machine. The results from these experiments were analyzed using the “Minitab” software package using either the two-sample t-test or the one-way analysis of variance. Unless stated otherwise, the significance of the samples coming from the same population was better than p ) 0.0005. All samples were of 5 replicates. Results After sample application, excess surface moisture was removed by blotting, followed by the reaction and a short washing step to remove excess reagents and buffer salts. To investigate the water uptake properties, the tanned paper was wetted and dried several times, so it is possible that protein was removed, thus giving false conclusions with respect to the amount of protein immobilized by tanning. Accordingly, total nitrogen of paper which had been treated with BSA, 2% catechol and tyrosinase was estimated before washing (1.1025% ( 0.094) and after five cycles of washing and drying (1.14% ( 0.062). Total nitrogen of paper before treatment was 0.1375% ( 0.062. It appears therefore from the nitrogen assay that although about 20 mg of protein was applied to the paper in solution, the addition of other reagents and subsequent blotting removed nearly two-thirds of this protein, leaving about 8 mg of BSA per paper strip. However, once this protein had been tanned it became resistant to washing and drying. There was no sign of any loss of nitrogen after five washes, so we deduce that the protein was immobilized within the cellulose matrix by the tanning process. In the first series of experiments, treatment of paper with DGG or tyrosinase on their own was shown to have no effect, but the treatment of DGG with tyrosinase gave a very significant reduction in water-holding capacity of the paper (Figure 1). If the DGG is polymerized with EDC but not tanned, significantly less water was absorbed than when only DGG was added, but the effect was not so great as when the DGG was tanned with tyrosinase, and tanning the coupled DGG did not increase its water resistance. The rest of the experiments constituted the second series, using the Whatman chromatography paper. To check further that the system was working as a mimic of a catechol tanning system and that the tyrosinase had to oxidize the catechol for the system to work, the enzyme was inhibited. With tropolone added to paper (Whatman) treated with BSA, catechol and tyrosinase, the wetted and blotted paper held 1.4 ( 0.013 g‚g-1 of water, as against the control value (no tropolone) of 0.72 ( 0.0014 g‚g-1. This shows that the system is working as a model of the tanning system and that the enzyme is sufficiently active at the concentrations of reagents higher than would normally be used in solution. Wetting and drying the treated paper decreased the water retention, the decrease was more marked as the number of
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cycles increased (Figure 2). Also the quantity of protein used was important (Figure 2); 15 µg of protein impregnated into the paper was not as effective in decreasing water retention as was 20 µg. Other proteins and polylysine were also used as substrates. Casein responded very poorly, showing some effect of waterproofing after tanning, but a drop in water retention of only 10% in the tanned samples as opposed to the halving of water retention in paper using BSA as the substrate (Figure 1). Polylysine waterproofed the paper as well as BSA (down to 0.55 ( 0.06 g‚g-1 after four cycles of wetting and drying). Gelatin, even when untanned, showed some waterproofing (Figure 3), but when tanned reduced water retention as effectively as BSA. However, untanned gelatin presented hardly any barrier to water when the impregnated paper was soaked for a long time. Water retention with untanned gelatin was 1.82 ( 0.11 g‚g-1 after 2 h soaking, whereas gelatin tanned with 2% catechol reduced the water retention to 1.167 ( 0.12 g‚g-1. Tanned BSA seemed more resistant to water, however, and after several cycles of wetting and drying retained only 0.77 ( 0.074 g‚g-1 of water after a 4 h immersion (Figure 4). Several tanning agents were used. Catechol was chosen as the standard since it seemed most effective. Phenol was as good with gelatin (Figure 3), and tyrosine, DOPA and DOPamine were almost as good with BSA, but N-acetyldopamine was not very effective. Experiments on the mechanical properties showed the stiffness of the paper to be a direct function of the water content. All the dry papers had the same modulus of about 5 GPa. However, the only tests were tensile, which is a rather crude way of measuring mechanical interactions between fibers within a composite material. The major difference detected was between fully wet untanned paper (about 1 g‚g-1 water, mean modulus 1.524 MPa) and the fully wet tanned paper (about 0.5 g‚g-1 water, mean modulus 58.25 MPa). Discussion When they are tanned, proteins not only became dryer and stiffer, they also darkened. This is due to polymerization products of the catechols forming structures that share some common properties with respect to hydrophobicity and hydrogen bonding capacity to the vegetable tannins or lignin. In general there was no correlation between waterproofing and color, which is consistent with the observation that some cuticles of insects are stiffened and dry in the absence of coloration.17 The experiments described here show clearly that it is possible to model the essential elements of tanning, as occurs in many invertebrates, but notably the insects. In our system, the paper is providing an inert framework (which could be likened to the chitin microfibrils in insect cuticle), but the reactions are taking place in concentrated solution. The addition of relatively small amounts of the tripeptide, DOPAGly-Gly, which is subsequently tanned, can very significantly increase the hydrophobicity of a paper substrate with associated changes in stiffness. This is an effective model of the mussel byssus adhesive where the phenolic residue
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(DOPA) is present in the protein phase before the phenoloxidase is added.3,18 An even greater effect can be achieved if the paper is impregnated with bovine serum albumin or other protein which is subsequently tanned with a phenolic material which is then oxidized. This mimics more closely the system in insect cuticle and appropriate structures in other invertebrates where the protein matrix is laid down over a period of days and tanned in bulk. Surprisingly the degree of water retention is not reduced more by using a hydrophobic protein (we used casein) which may be related to the accessibility of the interior of the protein to a water-borne tanning agent. Perhaps the casein could not be properly tanned. Wetting and drying the tanned material causes a reduction in the amount of water retained; this was also noted in the isolated cuticle of Calliphora erythrocephala where even untanned cuticle became less hydratable as the number of cycles increased, possibly due to the generation of beta structure in the protein.10 Catechol tanning of insect cuticle results in a significant degree of hydrophobicity, independent of any proposed phenolic cross-links, and was mooted by Vincent and Hillerton.8 Although this concept was vigorously opposed at the time, it has now become a central part of the theory of cuticular tanning19 and, by extension, of catechol tanning in general. Acknowledgment. We thank CIBA-Geigy for an ACE award for collaboration in Europe. M.G.P. acknowledges
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funding by the Deutsche Forschungsgemeinschaft and by the Fonds der Chemischen Industrie. References and Notes (1) Andersen, S. O. Cuticular sclerotization. In Cuticle Techniques in Arthropods; Miller, T. A., Ed.; Springer-Verlag: Heidelberg, Germany, 1980. (2) Andersen, S. O. Sclerotization and tanning of the cuticle. In ComparatiVe Insect Physiology Biochemistry and Pharmacology, Kerkut, G. A., Gilbert, L. I., Eds.; Pergamon: Oxford, U.K., 1985; Vol. 3, p 59. (3) Waite, J. H. Comp. Biochem. Physiol. 1990, 97B, 19. (4) Peter, M. G. Angew. Chem., Int. Ed. Engl. 1989, 28, 555. (5) Peter, M. G. Chem. Unserer Zeit. 1993, 4, 189. (6) Andersen, S. O.; Peter, M. G.; Roepstorff, P. Comp. Biochem. Physiol., B 1996, 113, 689. (7) Pryor, M. G. M. Proc. R. Soc. London, B 1940, 128, 378. (8) Vincent, J. F. V.; Hillerton, J. E. J. Insect Physiol. 1979, 25, 653. (9) Fraenkel, G.; Rudall, K. M. Proc. R. Soc. London 1940, 129, 1. (10) Vincent, J. F. V.; Ablett, S. J. Insect Physiol. 1987, 33, 973. (11) Vincent, J. F. V. The mechanical properties of biological materials. Symp. Soc. Exp. Biol. 1980, 34, 183 CUP. (12) Andersen, S. O.; Jacobsen, J. P.; Roepstorff, P.; Peter, M. G. Tetrahedron Lett. 1991, 32, 4287. (13) Peter, M. G.; Andersen, S. O.; Miessner, M.; Hartmann, R.; Roepstorff, P. Tetrahedron 1992, 48, 8927. (14) Schaeffer, J.; Kramer, K. J.; Farbow, J. R.; Jacob, G. S.; Stejskal, E. O.; Hopkins, T. L.; Speirs, R. D. Science 1987, 235, 1200. (15) Koeppe, J. K.; Mills, R. R. Insect Biochem. 1975, 5, 399. (16) Kahn, V.; Andrawis, A. Phytochemistry 1985, 24, 9065. (17) Andersen, S. O. Nature 1974, 251, 507. (18) Rzepecki, L. M.; Nagafuchi, T.; Waite, J. H. Arch. Biochem. Biophys. 1991, 285, 17. (19) Andersen, S. O.; Hojrup, P.; Roepstorff, P. Insect Biochem. Mol. Biol. 1995, 25, 153.
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