Marine Metal Chelating Proteins - ACS Symposium Series (ACS

Chapter 20

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Marine Metal Chelating Proteins Douglas C. Hansen and J. Herbert Waite College of Marine Studies, University of Delaware, Lewes, DE 19958-1298 The attachment strategy of the common sea mussel Mytilus edulis relies on the formation of byssal threads which originate in the animal at one end and attach to a surface at the other. The point of attachment for these threads are small plaques which contain an adhesive protein. This protein contains the amino acid 3,4-dihydroxyphenylalanine (DOPA). DOPAcontaining proteins have been isolated from other mytilid bivalves, namely Mytilus californianus and Geukensia demissa. Being an o-diphenol, this amino acid enables the adhesive plaque to displace water molecules through hydrogen bonding and to bind to metal ions that are present in the substrata. This chemisorption effectively anchors the mussel to the surface and allows it to resist the physical forces to which it is exposed. The byssal threads of these bivalves have demonstrated preferential accumulation and sequestration of trace metals. The adhesive qualities and the metal binding properties of this protein in aqueous environments present today's researcher with numerous potential uses and applications. The common sea mussel Mytilus edulis has an attachment strategy that relies on the formation of byssal threads which originate in the animal at one end and attach to a surface at the other. The ability of mytilid bivalves to attach themselves to objects underwater has been an object of curious fascination for man for centuries. Indeed, Aristotle mused that byssal threads were some sort of parasite or symbiotic plant that grew into the animal (1). These byssal threads are actually extracorporeal tendons that originate in the byssal retractor muscles and attach to any object that the animal can find (2). Mussels, like barnacles, are adhesive opportunists, and these animals will attach themselves to anything that provides them with a secure, solid holdfast. The points of attachment for these threads are small, flattened ovoid discs, or plaques, which contain the adhesive protein. The threads fuse with the plaque in such a way as to increase the surface area with the plaque material (2). 0097-6156/91/0444-0256$06.00/0 © 1991 American Chemical Society In Surface Reactive Peptides and Polymers; Sikes, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


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The formation of the thread and plaque is the result of a very complicated secretory process that is orchestrated by the foot of the mussel. This organ is very muscular and is covered with cilia (3). On its ventral surface, the foot has a groove extending from the base to the tip where it ends in a distal depression. Byssal threads are formed in the following sequence of events. The valves of the animal gape and the foot emerges. After a brief exploratory interval, the foot pushes the distal depression against a surface until the bulk of the water is displaced (4). Then, using a series of contracting muscles, the ceiling of the depression is lifted creating a vacuum within. Adhesive protein is discharged into the evacuated cavity through a series of 'injector ports' (4). After a few minutes the foot retracts and, as it does, the plaque emerges from the distal depression and the thread from the ventral groove to form a contiguous attachment structure. When the thread has completely disengaged from the groove, the sequence is ready to be repeated. At the outset the thread and plaque are a milky-white color, but over time they take on a golden-brown luster. This is evidence of the sclerotization that is taking place (5). The foot has a number of exocrine glands, which produce the various chemical components of the byssal thread. These are the phenol gland, the collagen gland and the accessory gland. The phenol gland is responsible for secreting the adhesive protein at the interface; the collagen gland secretes the tensile element present in the core of the threads, and the accessory gland secretes the coating that forms the cortex surrounding the thread (2). The protein that is present in the plaques and also in the cortex of the thread is the cornerstone of the adhesive strategy of these organisms. Isolation and Characterization of Adhesive Protein Simple hydrolysis of the byssus of M. edulis yields an amino acid composition that is high in glycine and lower in hydroxyproline and proline for the whole thread, as expected for a structural, fibrous protein complex (6). A similar composition is found in the plaques except that there is a higher amount of L-dopa present. This amino acid is named dopa from the German nomenclature "dioxyphenylalanin" and is a post-translational modification of tyrosine. Intuitively, the existence of a precursor protein containing this amino acid was suggested, and an assay to facilitate its location sought. In 1937, L.E. Arnow (7) described an assay for the colorimetric determination of the components of 3,4dihydroxyphenylalanine and tyrosine mixtures. This assay, despite its age, is very specific and yields a dinitrocatechol that absorbs strongly at 500 nanometers. With this assay then, it was quite easy to demonstrate that the adhesive, or dopa-containing, protein was indeed present in extracts of the foot, thread and plaque (8). Extraction of the dopa protein from the tip of the foot yields a pure protein as determined by acidic polyacrylamide gel electrophoresis (8). The amino acid composition of this purified dopa protein contains hydroxyproline, although it

In Surface Reactive Peptides and Polymers; Sikes, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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is not collagen as previously suspected due to its low glycine content and high levels of hydroxylated amino acids, such as dopa and lysine. The molecular weight of this protein as determined by polyacrylamide gel electrophoresis in the presence of cetylpyridinium bromide is 130,000 (± 10,000) and it has a pi above 10 (9). Tryptic digestion of the protein yields a consensus decapeptide that is


1

2

3

4

5

6

7

8

9

10

NH2 Ala Lys Pro Ser Tyr Hyp Hyp Thr Dopa Lys COOH (Dopa) and is repeated approximately 80 times (10). Researchers at Genex Corp. (Π) and at Boston University (Laursen and Connors, unpublished studies) have sequenced about 80% of a cDNA prepared from dopa-protein mRNA and have found 76 repeats of either hexa- or deca-peptides with many of the positions being highly or completely conserved therefore corroborating the repeat structure predicted earlier for this protein. The dopa protein isolated from other mytilid bivalves, namely Mytilus californianus and Geukensia demissa, have similar amino acid sequences in their peptides. Consensus decapeptides of M. californianus and G. demissa are, respectively: 1

2

3

4

5

6

7

8

9

10

NH2 Ile Thr Tyr Hyp Hyp Thr Dopa Lys Hyp Lys COOH (Dopa)

1

2

3

4

5

and 6 7

8

9

10

NH2 Gin Thr Gly Tyr Ser* Ala** Gly Dopa Lys (Dopa) *(Val/Asp)**(Pro/Hyp)

COOH

(12,13). The similarity between M. edulis protein and M. californianus proteins is quite striking. Positions 2 to 8 of the M. californianus decapeptide are essentially indistinguishable from M. edulis decapeptide positions 4 to 10 (12). Comparing the M. edulis consensus peptide to that of G. demissa, it is clear that there is a superimposibility of the second dopa and lysine residues. In all three cases, dopa/tyrosine positions are found three residues apart and the second dopa/ tyrosine residue, located at the carboxy end, is followed by a lysine. This illustrates the continuity that seems to be present in the adhesive proteins of mytilid bivalves. The secondary structure of this polypeptide in solution has yet to be elucidated. It has a CD spectrum that is characteristic of a random coil, either in the presence


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of sea water-like conditions or in 6M guanidine hydrochloride. To date, laboratory results indicate that when a peptide consisting of 20-30 consensus repeats is exposed to ascorbate, oxygen and mushroom tyrosinase at a pH of 7.2, only the second tyrosine of each consensus repeat was hydroxylated 100%. This clearly suggests that the conformation of the polypeptide is not a random coil, but that tyrosines particularly in position 5 are sheltered possibly by a /}-turn (14).


Adhesion and Complexation of Metals o-Diphenols form hydrogen bonds with hydrogen acceptors that are more powerful than water so displacement of water molecules or dehydration of adsorbent surfaces can occur (15). This phenomenon is well known in the technology of tanning of animal skins where the use of polyphenols vegetable tannins is applied to cure the collagen present in the skin to produce leather (16). One of the actions of the mussel adhesive in sticking to a film that is made out of a protein or carbohydrate (as in the case of organic films that form on surfaces in seawater) would be to locally dehydrate the surface thereby displacing the water (2). Another property is the ability to chelate metals (17). The bonds between the oxygens present in the quinone at ring positions 3 and 4 and a metal (iron in this case) are very short and energetically favorable (18). There are three stability constants in the chelation of iron (III) by o-diphenols, with log K values of 20, 15, 10, and these are associated with mono-, bis- and tris-complexes, respectively (19). These complexes result in three distinct colors - the mono-complex being a green color in solution, the bis- resulting in a blue and the tris- in a red color. The mono-complex occurs at an optimum pH of 5.0, the bis at 7.0 and 9.5-10.0 for the tris-complex (20). Green and blue iron complexes are formed in vitro by the mussel protein, but the stability of these is hard to assess due to the insolubility and multifunctionality of each molecule (Waite, unpublished results). The question remains as to whether the complex that is being formed is between phenolic residues on two different molecules or whether there is a bend in the protein and an ion is shared by two different residues of the same chain. o-Diphenols also have a high cumulative stability constants for other metals such as aluminum, zinc, titanium oxide, silicon dioxide and manganese and iron oxides (19,21,22). McBride (22) has demonstrated that catechol and hydroquinone are indeed adsorbed and oxidized by iron and manganese oxides. This chemisorption has also been demonstrated on aluminum oxide containing minerals such as gibbsite and boehmite and is dependent on their crystal structure (23). Environmental toxicologists have been using mussel byssus as a bioindicator of chemical contamination since the 1970's. Contaminants accumulated in the byssus of M. edulis include halogens and radionuclides such as U, Pu, Po and Am (24,25,26), arsenic (27) and transition metals including Cu, Co, Zn Ti, Mn and V (28,29,30,31). That these metals are strongly bound in byssus is suggested by the fact that they remain bound even after extensive washes with IN HCI or s


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IN NaOH (29). There is controversy with respect to how these metals are deposited in the byssus. The presence of metals in M. edulis byssus greatly exceeds the concentration of metals in seawater by a factor of 10 to 10 (29). Tateda and Kayanagi (31) have shown that when the bifurcate mussel, Septifer virgatus and M. edulis share the same seawater, S. virgatus seems to preferentially accumulate Co and Mn whereas M. edulis accumulates Fe and Zn. If the adhesive protein of these two mussels is largely responsible for the metal binding, then protein sequence and secondary structure may play a role in determining the metal ion-affinity of various dopa-containing proteins. Why would an animal make a polymer that has such high affinities for so many different metals? Although there is, of course, no pat answer to this, mussels are unquestionably adhesive opportunists. It is therefore no accident that three of the most abundant metals in the earth's crust, Si, Al and Fe (32) are precisely those for which the o-diphenolic moieties show their highest affinity.


4

6

Potential Uses and Applications The mussel protein adhesive qualities and its metal binding properties in aqueous environments present today's researcher with numerous potential uses and applications. It's not surprising that mans' attention should turn to agents in the natural environment that routinely adhere to surfaces in the presence of water. Adhesive technologists go to great lengths and expense to prepare surfaces and seal joints to protect them from moisture. An organic adhesive that adheres and cures in environments such as the mouth, orthopaedic implants and underwater construction is highly sought. This niche may be filled in the future by the dopacontaining bioadhesives. A good bioadhesive spreads spontaneously on its substrate, especially when adhesion is derived largely from van der Waals dispersion forces between the two materials. Good spreading means that the surface tension of the adhesive is lower than that of the adherend. In the marine mussels, the adhesive is always in direct competition with seawater for a particular surface. On non-polar surfaces, water interacts through weak dispersive forces. Since the adhesive protein is larger than a molecule of water, it offers the surface more dispersive interactions per molecule than does water, so the water is easily displaced (33). When the mussel is exposed to polar vs. non-polar substrates such as slate and teflon, the mussel will "select" the slate over the teflon to attach itself. If the mussel is only offered teflon, it will attach itself to that as well (33). On slate, however, due to its high surface energy with abundant charged polar groups, water tends to interact strongly through hydration spheres, hydrogen bonding, dipoles and induced dipoles as well as dispersive forces. Due to the strength of these interactions, the mussel cannot hope to spontaneously push aside the water with its adhesive protein. It can, however, establish a foothold that is fortified with interactions more energetic than the best that water has to offer, thus the higher bonding strength (4). The high catechol-Fe stability constants suggest that the proteins may have potential



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as organic anticorrosive compounds in aqueous environments. This idea is supported by a number of commercial rust-proofing procedures that currently involve addition of catechols to coating resins (34-38). The catechols may work as inhibitors by forming an adsorbed layer on the metal surface by means of the constituent hydroxyl groups. The advantage of using large natural proteins with catecholic groups is compelling. First, proteins are less soluble than the low molecular weight catechols presently in industrial use, and therefore are more likely to stay where they are deposited. Secondly, the mussel glue proteins have multiple (up to 150) catecholic groups per molecule. The stability of the proteinmetal interface is much higher than the stability of the single catechol:metal complex. This is reinforced by the finding that the degree of corrosion inhibition for iron in IN HCI varied systematically with chain length of the inhibiting molecule (39). Thirdly, the mussel glue protein can be easily imitated by enzymatic modification of genetically engineered analogues (40). It is well known that the mussel glue penetrates to the bare metal in making its adhesive bond to the metal surface (41). Combining this fact with the high stability constants of the catechol: Fe system, some anticorrosive properties of the mussel glue may be involved in reducing corrosion rates of steel surfaces exposed to seawater. Another potential use of the metal-binding properties of the mussel protein is the semi-specific sequestration of soluble metals from seawater. A microfibrous material containing the protein may be used to remove metals from aqueous systems. This area of potential research centers on the metal binding properties of the byssus and the possibility of imparting this property to other materials by grafting or coupling the mussel glue protein to other polymers such as cellulose, chitin, dextran, polyacrylamide or sulfonated polystyrene. Literature Cited 1. Van der Feen, P. J. Basteria 1949, 4, 66-71. 2. Waite, J. H. Biol. Rev. 1983, 58, 209-31. 3. Tamarin, Α.; Lewis, P.; Askey, J. J. Morph. 1976, 149, 199-222. 4. Waite, J. H. Int. J. Adhesion and Adhesives 1987, 7, 9-14. 5. Waite, J. H. In The Mollusca Vol. I. Hochachka, P. W.; Wilbur, Κ. M . Eds.; Academic Press: New York, 1983; pp 467-504. 6. Benedict, C. V.; Waite, J. H. J. of Morph. 1986, 189, 261-70. 7. Arnow, L. E. J. Biol. Chem. 1937, 118, 531-7. 8. Waite, J. H.; Tanzer, M . L. Science 1981, 212, 1038-40. 9. Waite, J. H. J. Biol. Chem. 1983, 258, 2911-5. 10. Waite, J. H.; Housley, T. J.; Tanzer, M . L. Biochemistry 1985, 24, 501014. 11. Strausberg, R. L.; Anderson, D. M.; Filipula, D.; Finkelman, M.; Link, R.; McCandliss, R.; Orndorff, S. Α.; Strausberg, S. L.; Wei, T. ACS Symp. Ser. 1989, 385, 453-64. 12. Waite, J. H. J. Comp. Physiol. 1986, 156, 491-6.



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13. Waite, J. H.; Hansen, D. C.; Little, Κ. T. J. Comp. Physiol. 1989, 159, 517-25. 14. Williams, T.; Marumo, K.; Waite, J. H.; Henkens, R. Arch. Biochem. Biophys. 1989, 269, 415-22. 15. Hagerman, A. E.; Butler, L. G. J. Biol. Chem. 1981, 256, 4494-7. 16. Bienkiewicz, K. In Physical Chemistry of Leathermaking, Krieger Pub. Comp.: Malabar, FL, 1983, pp 385-406. 17. Pierpont, C. G.; Buchanan, R. B. Coordination Chem. Rev. 1981, 38, 4587. 18. Raymond, K. N.; Isied, S. S.; Brown, L. D.; Fronczek, F. R.; Nibert, J. H. J. Am. Chem. Soc. 1976, 98, 1767. 19. Sillen, L. G.; Martell, A. E. Chem. Soc. London 1971, Suppl. No. 1, Spec. Publ., 25. 20. Hider, R. C.; Howlin, B.; Miller, J. R.; Mohd-Nor, A. R.; Silver, J. Inorg. Chim. Acta 1983, 80, 51-56. 21. Bartels, H. Helv. Chim. Acta. 1964, 47, 1605-9. 22. McBride, M . B. Soil Sci. Soc. Am. J. 1987, 51, 1466-72. 23. McBride, M . B.; Wesselink, L. J. Env. Sci. Tech. 1988, 22, 703-8. 24. Koide, M . ; Lee, D. S.; Goldberg, E. D. Est. Coast. Shelf. Sci. 1982, 15, 679-95. 25. Hamilton, Ε. I. Mar. Ecol. Prog. Ser. 1980, 2, 61-73. 26. Hodge, V. F.; Koide, M.; Goldberg, E. D. Nature 1979, 277, 206-9. 27. Unlu, M . Y.; Fowler, S. W. Mar. Biol. 1979, 57, 209-19. 28. Pentreath, R. J. J. Mar. Biol. Assoc. U.K. 1973, 53, 127-43. 29. Coombs, T. L.; Keller, P. J. Aquat. Toxicol. 1981, 1, 291-300. 30. Coulon, J.; Trutchet, M . ; Martoja, R. Ann. Inst. Ocean. Paris 1987, 63, 89-100. 31. Tateda, Y.; Kayanagi, T. Bull. Jap. Soc. Fish. 1986, 52, 2019-26. 32. Rochow, E. G. Silicon and Silicones. 1987, Springer-Verlag: Berlin. 33. Crisp, D. J.; Walker, G.; Young, G. Α.; Yule, A. B. J. Colloid and Interface Sci. 1985, 104, 40-50. 34. Faulkner, R. N.; O'Neill, L. A. S.C.I. Monograph 1964, 26, 177-88. 35. Pizzi, A. J. Appl. Polym. Sci. 1979, 24, 1247-55. 36. Toyota Motor Company. Rustproofing coating material, Japan Patent 57 139155. 1982. 37. Vacchini, D. Anti-Corros. Methods Mater. 1985, 32, 9-11. 38. Tury, B.; John, G. R.; Scovell, E. G. Corrosion Inhibition. Eur. Patent 239288. 1987. 39. Carrol, M . J. B.; Travis, Κ. E.; Noggle, J. H. Corrosion 1975, 31, 1237. 40. Marumo, K.; Waite, J. H. Biochem. et Biophys. Acta 1986, 872, 98-103. 41. Selin, H. H. Biologiya Morya 1980, 3, 97-9. RECEIVED August 27, 1990


Marine Metal Chelating Proteins - ACS Symposium Series (ACS

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