Surface Reactive Peptides and Polymers - ACS Publications

Chapter 24

Downloaded by CORNELL UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: November 26, 1991 | doi: 10.1021/bk-1991-0444.ch024

Some Structural and Functional Properties of a Possible Organic Matrix from the Frustules of the Freshwater Diatom Cyclotella meneghiniana D. M. Swift and A. P. Wheeler Department of Biological Sciences, Clemson University, Clemson, SC 29634-1903 Organic materials extracted from frustules of the diatom Cyclotella meneghiniana exhibit some characteristics common to calcium-biomineral organic matrices. Isolated siliceous frustules cleaned with 5% NaOCl, pH 8, were dissolved in an NH / HF solution. The remaining organic materials contained large portions of protein, some carbohydrate, and lesser quantities of phosphate and sulfate. The protein(s) in the aqueous soluble fraction contained 20% serine, 24% glycine and 10% aspartic acid. The extracted materials at concentrations up to 1 μΜ had no effect on in vitro SiO polymerization in solution, but did exhibit strong inhibitory capacities against in vitro CaCO crystallization at approximately 10 μΜ. This level of activity was roughly the same as for proteins obtained from carbonate biomineral indicating that the siliceous proteins, like the carbonate proteins, may contain highly anionic domains of polyaspartic acid or polyphosphoserine. 4

2

-2

3

Biosilicification has been studied far more intensely in diatoms than in any other group of organisms that utilize biosilica. Morphological studies of the elaborate diatom shells or frustules date from the 1800's. More recently, investigations have focused on the silicification process as reviewed by Sullivan (1). From these studies it is evident that the frustule is produced within a silica deposition vesicle (SDV) which is delimited by a membrane, the silicalemma. The SDV is formed adjacent to the newly formed plasmalemma after cytokinesis. There is evidence that in some species the SDV is formed by coalescence of small vesicles of unknown origin. The Golgi has been found in close proximity to the SDV and is considered a likely source of the vesicles. Silica appears to be deposited centripetally within the SDV shortly after its appearance (2). Temporal patterns of uptake and incorporation of silica by diatoms have been characterized (1) and silica ionophores have been identified and localized (3). 0097-6156/91/0444-0340$06.00/0 0) at 1800 χ g for 2 min (4). Further purification of the frustule 5

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


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SURFACE REACTIVE PEPTIDES AND POLYMERS

fraction was achieved by washing it 2-3 times for 15 minutes in 100% dimethylformamide (DMF). This step removed pigments still detectable in the frustule fraction after the water washes. Frustules were collected and washed once more with dH20. This final wash was followed by dialysis to remove any remaining DMF. Frustules were then frozen and lyophilized and the weight of the silica plus organic material determined. In addition, after the lyophilization, some material was treated for 3-5 minutes with 5% NaOCl, 10 mM Tris, pH 8.0 followed by centrifugation and washing with dH20. Frustules were then dialyzed, lyophilized and weighed. The removal of the organic casing from the mature frustules was determined by exposing dried samples to ruthenium red stain and examining them using light microscopy. The casing contains mucopolysaccharides known to stain specifically with ruthenium red (2,5). Lack of staining was taken to indicate that the organic casing had been removed. When the non-NaOCl cleaned (untreated) frustules were exposed to ruthenium red they stained intensely, suggesting there was still some contamination from the organic casing or cell contents. However, the NaOCl cleaned (treated) frustules exhibited no staining. To dissolve the silica from the frustules, both the untreated and treated samples were then suspended in a solution of 1% HF and 3% NH4F, pH 5 for 48 hours at 4°C with constant shaking. The remaining organic material was then dialyzed exhaustively, lyophilized and weighed. The dried material was suspended in dH20 for 48 hours with constant stirring followed by centrifugation at 24,000 χ g for 20 minutes. The supernatant and pellet were collected separately, lyophilized and weighed. Fractions from treated frustules will be designated as supernatant (T) and pellet (T) whereas untreated fractions will be followed by (NT). A variety of compounds were exposed to the same extraction procedure to determine the effect of the dissolution medium on molecular size and structure. The test molecules included cytochrome C, bovine serum albumin, blue dextran, pepsin, and myoglobin. The dissolution method was deemed not especially deleterious to structure as both cytochrome C and myoglobin retained their heme groups and all retained their visible and ultraviolet absorbance profiles. All appeared to retain their approximate molecular sizes as well. Chemical analyses were performed on the supernatant fraction which was always resuspended in dH20 at 1 mg/ ml by dry weight. The pellet material, when analyzed, was suspended and sonicated to achieve a uniform suspension. Protein content was determined by the Miller (6) modification of the Lowry assay and carbohydrate by the Dubois et al. (7) method. Phosphate and sulfate analyses were performed on dried aliquots of both soluble and insoluble materials. Phosphate was determined by the Wheeler and Harrison (8) modification of the Marsh (9) method and sulfate by a modified method of Wainer and Koch (10). For this latter analysis, samples were oxidized in 30% H2O2 at 70 °C until all liquid was evaporated. Oxidized samples and standards resuspended in 0.4 ml of dH20 were vortexed with 0.1 ml of 0.1 M sodium acetate-acetic acid buffer. An ethanol (95%) suspension containing 3 mg of barium chloranilate in 0.5 ml was added and the samples vortexed about once per minute for 10 minutes. After centrifugation, 0.5 ml



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Frustules of a Freshwater Diatom 343

of the supernatant was mixed with 0.2 ml of a 1:1 solution of glacial acetic acid:water and the absorbance of the resulting solution was determined at 530 nm. Extracts were also tested for an effect on in vitro silica polymerization rates using a modification of the method of Weres et al. (Π). In this assay, the rate of polymerization of silicic acid is determined by following the rate of disappearance of free silica monomers from a supersaturated solution. Free silica concentrations were determined using the beta silicomolybdate reaction (12). In this assay, silica monomers, short chain linear oligomers and perhaps even some small cyclic species will react with molybdic acid in less than 5 minutes and are termed molybdate active silica (MAS) (13). The pH of the polymerizing solution was maintained at 7 with BES buffer (163 mM). The initial silica concentration was 750 ppm as S1O2 (12.5 mM). The reaction was initiated by the addition of S1O2 and the pH was adjusted rapidly (3 crystallization rates was determined using the pH-stat system as described in Wheeler and Sikes (14). Molecular weights of the soluble materials were determined by gel filtration chromatography using HPLC and a TSK-30 column with a mobile phase of 0.05 M tris-acetate, pH 7.4. Amino acid analyses were performed using the method of Waite and Benedict (15) on a Beckman 6300 Autoanalyser. All dialyses were done using SpectraPor 3 membranes (3500 MWCO) at 4°C against dH2U with constant stirring. Results The results of the chemical analyses are shown in Table I. The relative amount of protein detectable following NaOCl treatment decreases in both the supernatant and pellet fractions. The supernatant (NT) has a higher proportion of protein when compared to carbohydrate than does the supernatant (T). The pellet has more detectable protein than carbohydrate from both the untreated and treated samples. Both supernatant extracts have roughly the same relative amount of phosphate, whereas both pellet fractions contain little detectable phosphate. Both pellet fractions contained small amounts of sulfate, with about twice as much appearing in the pellet (NT) as the pellet (T). Amino acid analyses of soluble and insoluble fractions from both untreated and treated frustules are given in Table II. Also included is the amino acid composition of cell contents collected from the supernatant of the first centrifugation after cells were sonicated. The most prominent amino acids in the supernatant fractions were glycine and serine, which for the treated supernatant were present at approximately two times the concentrations found in the cell contents fractions. Additionally, there were larger relative quantities of hydrophobic amino acids found in the pellet fractions than the supernatant fractions.


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Table I. Chemical Composition of Fractions Extracted from Frustules Fraction

2

Protein

Supernatant (NT) Pellet (NT) Supernatant (T) Pellet (T) 1


2

3

509 ± 307 ± 274 ± 190 ±

178 171 11 30

Carbohydrate 227 ± 63 ± 325 ± 60 ±

77 17 102 10

P0

S0

4

66±22 16 ± 12 73 ± 4 4± 2

η

4

6 3 3 3

3

n.d. 25 ± 8 n.d.3

14 ± 2

(jjgj mg dried wt.) Fractions designated Τ were obtained from 5% NaOCl cleaned frustules and fractions designated NT were from untreated frustules. n.d. not determined

Table II. Amino Acid Composition of Various Extract Fractions Amino Acid asp thr ser glu pro giy ala cys val met ile leu tyr phe his lys arg

Τ supernatant

Τ pellet

Cell Contents

NT supernatant

NT pellet

10.03 5.30 20.31 9.16 4.43 24.46 8.22 0.02 4.35 0.00 2.53 4.31 0.79 2.23 0.86 1.81 0.26

10.17 3.93 9.44 11.33 6.08 19.03 9.66 0.02 6.79 0.00 2.93 9.52 0.93 4.16 1.70 1.78 0.88

10.45 5.30 10.41 10.32 5.83 13.87 10.85 0.27 5.68 0.00 3.83 6.98 2.54 3.49 1.56 4.55 2.46

9.72 5.25 15.64 10.14 4.72 17.86 9.01 0.29 4.20 0.00 3.25 5.31 2.38 2.80 2.49 3.34 2.10

9.86 5.03 12.83 10.50 5.66 18.46 8.77 0.25 5.39 0.00 2.70 7.90 1.83 3.02 1.57 3.23 2.08

Analyses for degree of similarity of amino acid compositions were made using the method of Cornish-Bowden (16). In this method, a difference index between two compositions is determined according to the equation


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Frustules of a Freshwater Diatom 345 2

SAn=l/2Z(riiA-riiB)


t h

where I I J A or Π Ι Β is the quantity of the i type of amino acid contained in protein A or B. If SAn3 deposition. Failure to observe this may result from any of several possibilities. For example, the soluble materials tested thus far consisted only of non-NaOCl cleaned frustule extracts at a maximum concentration of 50 μg/πû. No effect was observed at concentrations of up to 0.5 mg/ml insoluble NT material. However, it is possible that there would be an inhibitory effect at higher doses. If these materials do have an effect on S1O2 polymerization rate, then this concentration may be below the level necessary to detect said effect. Also, it may be that the treated supernatant fraction might exhibit some effect on polymerization where the NT fraction does not. Other compounds tested in this assay that exhibited no effect on the rate of polymerization of silica include polyaspartic acid, copolymers of serine and aspartic acid, bovine serine albumin, polyacrylate, glycerol, and oyster soluble matrix. Borate did exhibit an inhibitory effect after initial silica binding, but at superstoichiometric levels (200mM). Meier and Dubin (27) also found that only stoichiometric levels of borate, of all the industrial compounds tested, produced an effect on the rate of silica polymerization. In this light, perhaps the lack of any measurable effect by the diatom extracts at the concentrations tested is not surprising. Because the concentration of free S1O2 at the site of polymerization is unknown, the concentration of S1O2 in the in vitro assays may be unrealistic as a model for the concentrations found in vivo. Also, if the materials extracted offer nucleation sites for deposition, then their function may be masked at the high rate of polymerization (hence, large number of nucleation events) occurring in the solution. Obviously, concentrations of extracts above those used thus far must be employed to test this idea. Also, various levels of S1O2 supersaturation should be evaluated. This process will be difficult to execute because of the small quantities of the soluble materials available. It is possible that the compounds extracted may interact with soluble or forming silica polymers only when immobilized on a substrate. This hypothesis is being explored. Additionally, the effect of a siliceous matrix may be to regulate the form or architecture of the silica deposited, with little or no effect on rate of deposition. Silica is known to occupy various morphs in plant hairs, the deposition of which correspond temporally with changes in organic components associated with the deposits (28). Finally, it is important to keep in mind that the mineralization process in diatoms is not one of crystallization but polymerization. There are no discrete surfaces formed, as the silica polymers exist in a substantially random network (18). At any point in


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time there are an unknown number of free silanol groups available for polymerization, not a fixed surface with discrete lattice sites as is found in growing calcium carbonate crystals. In conclusion, we have identified materials which are closely associated with the siliceous frustule, as evidenced by their resistance to attack by NaOCl, that have unique properties which suggest that they may have a role in biosilicification. The origin of these materials, their location in relation to the mineral, further physical characterization, and their in vivo function are all areas which are being explored.


Acknowledgments We gratefully acknowledge Dr. Herbert Waite for performing the amino acid analyses and Dr. Susan Kilham for providing starter cultures of the diatom. This work was funded in part by a grant from the South Carolina Sea Grant Consortium (APW) and a grant-in-aid of research from Sigma Xi (DMS). Literature Cited 1. Sullivan, C. W. In Silicon Biochemistry; Evered, D.; O'Conner, M., Eds.; John Wiley and Sons: N.Y., 1986; pp 59-89. 2. Schmidt, A - M . M.; Borowitzka, Μ. Α.; Volcani, Β. E. In Cytomorphogenesis in Plants; Werner, D., Ed.; 1981; pp 63-97. 3. Bhattacharyya, P.; Volcani, Β. E. Biochem. Biophys. Res. Comm. 1983, 114(1), 365-72. 4. Hecky, R. E.; Mopper, K.; Kilham, P.; Degens, Ε. T. Marine Biology 1973, 19, 323-31. 5. Duke, E. L.; Reimann, B. E. F. In The Biology of Diatoms; Werner, D., Ed.; Univ. of California Press: Berkeley, 1977; pp 65-109. 6. Miller, G. L. Anal. Chem. 1959, 31, 964. 7. Dubois, M.; Gilles, Κ.; Hamilton, J. K.; Rebers, P. Α.; Smith, F. Anal. Chem. 1956, 28(3), 350-6. 8. Wheeler, A. P.; Harrison, E. W. Comp. Biochem. Physiol. 1982, 71B, 62936. 9. Marsh, Β. B. Biochem. Biophys. Acta 1959, 32, 351-61. 10. Wainer, Α.; Koch, A. Anal. Biochem. 1962, 3, 457-61. 11. Weres, O.; Yee, Α.; Tsao, L. J. Coll. Inter. Sci. 1981, 84(2), 379-402. 12. Iler, R. K. The Chemistry of Silica. Wiley-Interscience: N.Y., 1979; ρ 97. 13. Marsh, A. R.; Klein, G.; Vermeulen, T. Report LBL-4415, 1975; Lawrence Berkeley Laboratory. 14. Wheeler, A. P.; Sikes, C. S. Am. Zool. 1984, 24, 933-44. 15. Waite, H.; Benedict, C. V. Meth. Enz. 1984, 107, 397-413.



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16. Cornish-Bowden, A. Meth. Enz. 1983, 91, 60-75. 17. Blank, G. S.; Robinson, D. H.; Sullivan, C. W. J. Phycol. 1986, 22, 382389. 18. Perry, C. C. In Biomineralization: Chemical and Biochemical Perspectives; Mann, S.; Webb, S.; Williams, R. J.P., Eds.; VCH: London, 1989; ρ 22356. 19. Coombs, J.; Volcani, B. Planta (Berl.) 1968, 80, 264-79. 20. Wheeler, A. P.; Rusenko, K. W.; Swift, D. M . ; Sikes, C. S. Mar. Biol. 1988, 98, 71-89. 21. Volcani, Β. E. In Silicon and Siliceous Structures in Biological Systems; Simpson, T. L.; Volcani, Β. E., Eds.; Springer-Verlag: N.Y., 1981; ρ 157200. 22. Rusenko, K. W.; Donachy, J. E. In Surface Reactive Peptides and Polymers: Discovery and Commercialization. Sikes, C. S.; Wheeler, A. P., Eds.; ACS Books: Washington, D.C., 1990. 23. Sikes, C. S.; Wheeler, A. P. CHEMTECH 1988, 620-6. 24. Wheeler, A. P.; Sikes, C. S. In Biomineralization: Chemical and Biochemical Perspectives; Mann, S.; Webb, S.; Williams, R. J. P., Eds., VCH: London, 1989; ρ 95-131. 25. Robinson, D. H.; Sullivan, C. W. TIΒS 1987, 12151-4. 26. Roth, R.; Werner, D. Z. Pflanzenphysiol. 1977, 83, 363-74. 27. Meier, D. Α.; Dubin, L. Corrosion, 1987, Paper 334. 28. Perry, C.C.;Wilcock, J. In Chemical Aspects of Regulation of Mineralization; Sikes, C. S.; Wheeler, A. P., Eds., University of South Alabama Publication Services: Mobile, AL, 1988; ρ 39-48. RECEIVED August 27, 1990


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