Biomineralization Principles and Applications in Bioinorganic Chemistry-V ~
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EI-lchlro Ochlal Junlata College, Huntlngdon, PA 16652 Oreanisms use some inoreanic material for the . nurnose " . of gaining mechanical strength and for some other minor purnoses. Examnles include "bone" and "dentine", the maior 'component i f which is a calcium phosphate mineral, A d "calcified shell" made of calcium carbonate. A group of marine algae called "diatoms" uses silica as its case (called "frustule"). These are typical examples of the so-called "biominerals". We would like to explore somewhat why such calcium and silicon compounds are used for these purposes by organisms and how these biominerals are formed. I t must be pointed out, however, that cellulose and its derivatives, such as chitin, i.e., some organic substances, are also used by many. organisms from bacteria and fungi to trees and insects . as wrapping material and outer shells that require some mechanical strength. We human beings, too, use inorganic minerals in order to build our shelters. The most commonly used materials for human shelter include wood, brick, and concrete (mixture of cement and sand). Except for wood, they are inorganicmaterial. Brick and cement are manufactured from inorganic material. They are used because of the ready availability (economic factor) of the raw materials and their properties suitable for the purpose. I t might be noted that these two reasons are of the same nature as "rule l" and "rule 2" expounded in order to explain bioselection of elements in the prkvious articles of this series ( 1 4 ) . (Four rules were delineated: rule 1 (called "rule of basic fitness or chemical suitability") assertsthat a certain chemical element is inherently (chemically) fit for a certain biological function, and rule 2 ("rule of abundance") suggests that organisms would have a preference for more abundant and readily available elements.) After all, the basic principles of our economic system of utilizing material would not be very much different from those an~iicableto oreanisms' bodilvuse. perhaps excent for the iss;; of "value" (&luding the aesthkiic value). Bricks are typically made of clay minerals, whose major component is aluminosilicates such as montmorillonite (Nao.33(All.e,Mg,33)Sia010(OH)znHz0). Cement, on the other hand, is made from limestone (CaC03) and clays or blast furnace slag containing aluminosilicate. A typical cement, Portland cement, is a mixture of 3CaO.SiO2, 2CaO-SiOn, and 3Ca0.AlnOl (plus other components) in varying proportions. ~and;whichis used tomake concrete in coniunction with cement, is made of essentially SiO2. It is to be noted that the major elements in these materials are calcium, silicon, and aluminum. Organisms utilize calcium and silicon as building material (biominerals) as mentioned above, but not aluminum. As a matter of fact, the nonessentiality of aluminum to organisms is rather a conspicuous exception to "rule 2" above. Although aluminum may eventually turn out to be essential to organisms in some rather minor functions, it is unlikely that aluminum, despite its abundance on theEarth, is required in the biosphere in significant quantity by organisms (see also ref 3). Certain trees. includinz Camellia sinensi (5) and S,mplocos spicoto (6),are kno& to accumulate aluminum. This fact mav implv either that thevabsorbaluminum inadvertently and toierke it or that al;minum bas a beneficial ~
effect on them. I t is not known whether aluminum constitutes some macrostructures that contribute to the mechanical strength of the trees.
Some B s r k Chemistry ot Calclum and Slllcon The dominant chemical species of calcium in aqueous medium is Ca(I1) cation. Because of its relatively large size (110-120 ppm), Ca(I1) forms insoluble compounds with relatively large anions such as C0s2-, C20p2-, and POa3-. Because of different dependences of lattice and hydration energy on the ionic radii, a simple salt would be more soluble as the ionic sizes of the cation and the anion differ more (7). That is, a salt of cation and anion of similar sizes tends to be less soluble. Ca(1I) binds referentially oxveen anions of acidic residues, particularl; of phosphaie, c&boxylate, and sulfate. Indeed, typical Ca(l1)-bindingproteins contain high levels of aspartaie and glutamate,-kd the carboxylaie groups of these amino acid residues are the major binding sites of Ca(I1) (8).Carboxylates and sulfates of carbohydrate derivatives (mucopolysaccharides) and phosphate of phospholipids would also bind Ca(I1). Ca(I1) also dehydrates easily in terms of both kinetic and thermodynamic senses because of a low charge-to-radius ratio, which determines the electron-withdrawing effect of a cation. In contrast, the dominant chemical species of silicon in aqueous medium is silicic acid, Si(OH)4 and its derivatives such as H3Si04-, HzSiOs, HeSi207, and H2Si205.These are believed to be the forms of silicon that are absorbed by organisms such as diatoms, sponges, and some plants. Their solubility in water is collectively of the order of 2 x M up to about pH 9; the solubility increases significantly above pH 9 (9). A characteristic feature of silicic acid is its tendency to condense and polymerize t o form gels (10, 11). (In contrast, Al(II1) is virtually insoluble between pH 5 and pH 11, above which it exists as A102- and its solubility increases.) The acidity of silicic acid is not particularly strong; its pK, is about 9.5 (10). Like other acids, it is capable of forming ester with alcohol (12): -Si-OH
-H,O + HOR + -Si-OR
(1)
However, this Si-0-C bond is relatively reactive and can be hydrolyzed relatively easily. Therefore, a commercial application of such a compound as the packing material based on silica (e.g., Cis column) for HPLC utilizes instead Si0-Si-R entities as shown in eq 2:
Calclum a s Bulldlng Mabrlal Calcium and its com~oundsfunction as cement as well as bulk material (of consfderable mechanical strength) in bioloeical svstems. Most cell membranes contain nlvco~roteins (01pep6doglycans), with its protein portion being'embedded in the membrane and its oliaosaccharide portion sticking out. These glycoproteins are belived to be involved in Volume 68 Number 8 August 1991
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cell-cell recognition and adhesion. In many cases, Ca(I1) seems to be required for the cell-cell adhesion (13). Ca(I1) is believed to bridge two negatively charged molecules, especially those containing carboxylates or phosphates, thus functioning as an adhesive between molecules. This effect is much more prominent with Ca(II), as it dehydrates more readily, than, e.g., with Mg(I1) (14). It must be pointed out, however, that this is not the only mode of c e k e l l adhesion (13). A component of the cell walls of plants is hemicellulose. Pectic substance, the major component of hemicellulose, contains polymers of galacturonic acid (see below), whose carboxylate groups bind Ca(I1). Here Ca(I1) is thought to ridge two molecules of pectic substances and contribute to the adhesion between adjacent plant cells.
COOH
t o
' 6H galacturonic acid
An unusually high level of bound silicon was detected in the arterial wall tissue, especially in the innermost layer (intima) (19). This may suggest that silicon is necessary for the integrity and stability of the arterial wall. A deficiency of silicon may cause disruption of the intergrity of the intima and, thus, trigger the deposition of chole&erol. Silicon, deposited as silica gel, is utilized by a number of oreanisms as case (frustule). .,scales. and soicules. Soonees. - .a group of marine invertebrates (phylum Porifera), contain some skeletonic structures in the soft bodies. They are either spicules made of CaC03 in some species, SiOz spicules in other suecies, or oreanic fibrous material called "s~oneinin" (as in ihe case ofbath sponge). The shapes and sizes of spicules are widely varied. Some chrysophytae species, single-celled algae, deposit silica gel plates on their cell surfaces. The most conspicuous and-important silica-bearing organisms are radiolarians (protozoa) and diatoms. Diatoms, photoautotrophs, are mokly marine and numerous (more than 10,000 species). Their cell walls consist of siliceous frustule encased in organic coating. The silica in these shells is similar in chemical composition to a silica gel, Si02.xH20. Planktonic orotozoans. radiolaria. use ooaline silica for their outer skeleions. The variation of their'shapes is extraordinarv (20). A minor number of radiolaria use strontium sulfatdins&ad of silica at the stage of isospore (earlier developmental stage). Silica is often deposited as amorphous silica gel in the , horsetails and masses shoots of vascular ~ l a n t s es~eciallv (21). Eguistrurn a & m (a-horsetail) has been shown to colla~sewhen mown on silica-free nutrient solutions but to be normally erict when sodium metasilicate was included in the growth medium (22).
-
The main components of cell membranes are phospholipids with the phosphate portion being exposed to aqueous media. The negative charges on the adjacent phospholipid molecules tend to destabilize the membrane structure. This repulsive effect seems to be compensated by cations, particularlv bv Ca(I1).There are anumber of exoerimental data to indicatethe stabilizing effects of Ca(I1) on membrane structures (15). As indicated above, Ca(I1) forms insoluble compounds with C032-. C?OL2-and PO$. Calcium carbonate is commonly used b; a-number of -invertebrates as shells, spines, and endo- and exoskeletons (e.g, as in coral) and is found in the egg shells of birds and reptiles. Coccolithopholids, marine algae, form CaC03 plates on their cell surfaces. The deposit of this CaC03 is believed to be a major source of the chalk bed. Calcium oxalate is found in most ferns and mosses and some algae, and occasionally found in insect eggs and larval cuticles. Calcium phosphate is the major component of bones and dentines of vertebrates. It is also found in some protozoa, coelenterates, arthropods, and brachiopods. Slllcon a s Bulldlng Malerlal It is only recently that silicon has begun to attract attention of biochemista; hence, silicon biochemistry (10, 16) is much less understood than that of calcium, but it seems to function in the two capacities as described above, i.e., as cement and building material. Schwartz (17,18) and Carlisle (19) found high levels of tightly bound silicon in many acidic mucopolysaccharides. Silicon was also found in polyuronides (uronide = carboxylate derivative of a carbohydrate) of plants, e.g, at 2580 ppm in pectin of citrus fruit and 450 ppm in alginic acid of horsetail kelp. It was reported (17) that the tightly bound silicon in these material was covalently linked to carbon through an oxygen atom, though no definite evidence was presented to support this assertion. Alternatively, bonding through hydrogen bond (11) may be important in these complexes (see structures 1 and 2 below). Neither of these structures has been verified. Silicon was established to be involved also in the formation of the mucopolysaccharides or glycosamineglycans of connective tissues and was found to be bound to the proteins in connective tissues, especially collagen (19). The nature of the function of silicon in these tissues is not well understood but could be crosslinking of the polymers through chemical bindings such as shown in 1 and 2. 828
Journal of Chemical Education
.
Biomlnerallzatlon The mechanisms of the formation of calcium- or siliconbearing macrostructures in biological systems (often called "biomineralization") are far from understood (23). The following is a brief outline of what is known about the subject, emphasizing the difference between calcium and silicon. The crystallization process is typically autocatalytic (i.e., once a nucleus is formed, crystallization proceeds rapidly). Thus the difficult stage is nucleation or seed formation. Therefore, research is directed mainly toward identifying the nucleation sites. Slllca Gel Fonnatlon In Dlatorns The amino acid composition of the organic coating was studied for several species (24). The protein in the cell wall contains more serine, threonine, and glycine and less glutamic acid, aspartic acid, aromatic amino acids, and sulfurcontaining amino acids than the proteins within the cell do. Serine and threonine both have aliphatic hydroxyl groups. This finding suggested a mechanism in which silicic acid
undergoes a condensation reaction (esterification in a sense) with the hydroxyl groups on the adjacent serine (or threonine) residues (24) (3).Alternatively, this interaction may he simply of the hydrogen honding type (4) (11):
The small aggregates of SiO, units formed are considered then to act as the nucleus for the crystal growth. Perhaps this mechanism is an oversimplification, hut it seems to represent the essential features of the siliceous layer formation in diatoms, nonetheless (23,25,26). Calcium Carbonate Shell In MoIIuscs Molluscan shells are generally composed of calcium carbonate (in either calcite or aragonite crystalline form) enclosed in an organic matrix. The organic matrix was observed to form prior to the mineralization and, hence, was thought to provide a crystal nucleation site. The major component of the organic matrix protein is a glycoprotein conininins .--------hieh - ~ - lev& of acidic amino acids and acidic mucopolysaccharides. The study of the amino acid compositions of- the elvconroteins from several soecies demonstrated the predominance of aspartate and giycine and the predominance (18-38% deoendine on the species) of the sequence A ~ ~ X ~ A (x'= S ~ -mos& X g~ycinebrserine) in the protein matrices associated with calcite (27.28). The corresponding associated &th ar; number is lower, %14% in the gonite (28). The problem of what factors determine the crystal structure (calcite vs. aragonite) is unsolved. I t was pronosed based on these observations that Asp-X-Asp-X acts as a template for the mineralization (23,291:
-. .
Bone/Dentin Formatlon Osteoblasts (cells responsible for hone formation) produce and secrete matrix proteins such as collagen as well as some special proteins. One such protein called "osteonectin", a phosphate-containing glycoprotein, hinds both Ca(I1) ions and apatite crystallites (apatite is the major mineral of hone) to collaaen and also facilitates the nucleation of calcium phosph&mineral oncollagen (28).It appears that thecalcification site is the suhmicroscopic vesicle called "matrix vesicle". Its memhrane derives from that of osteoblast hut contains more sphingomyelin and phosphatidylserine than the mother cell; a n d also contains ATPase, pyrophosphatase, and other phosphatases (31). Perhaps osteonectin is an integral part of thisvesicle, and the phosphatases provide the phosphate needed for the bone formation. The phosphate groups of the vesicle memhrane andlor of osteonectin (or the like) would provide the Ca(I1)-binding sites in the hone formation. The Ca(I1)-PO4 aggregates thus created serve as nuclei for the mineralization. Concludlng Remarks The nucleation sites for the hiominerals appear to he chemically compatible with the minerals: -C-OH (alcoholic hvdroxvl) for silica, -COO- for calcium carbonate and -1'0,'for Ea~riumphosphate (apatite),though this generalization needs to he confirmed yet in many syatems. If this generalization turns out to be true, this strongly suggests that the bio-usage of an element is indeed determined by its hasicchemical nature (rule 11))in thiscase. too. As for rule 2 - - ~ - - ~ (abundance rule), calcium aidsilicon, being among the most ahundant elements on the earth, meet the criterionsquarely. Similarly ahundant and likely candidates for the job would include Me. Al, and Fe. MdII), being smaller than Ca(Il),would notjorm appropriari insoluhl~compoundsas Cal.I I .I does. The insoluble compounds of the other progenies of Ca, i.e., of Sr (and maybe B& are utilized occasion~llyas mentioned earlier. Iron seems to constitute hiominerals (often as magnetite Fe304)in relatively minor ways (32). One serious problem with aluminum is its inaccessibility. Al(II1) forms insoluble species in aqueous media of ordinary pH (see above). Its small size and high electric charge makes AI(II1) an extremely strong acid, perhaps too strong an acid for biological system. These characteristics would make aluminum less desirable for hiological use than otherwise expected. Aluminum is known to he toxic, and aluminosilicate has recently been reported to deposit in the brain of Alzheimer disease patients (33). Silicon, here, is believed to function as a detoxifying agent against aluminum (34). I t may he pointed out that silicate particles, however, are a health hazard, manifested in the form of silicosis or ashestosis. ~
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Oehisi, E-I. J. Cham. Educ. 1978.55.631-633. Ochiai, E ~ IJ. . Chem. E d u c 1987.64.942-944. Oehiai. E-I. J. Chem. Educ. 1988.65.943-946, 0chisi.E-I. J.Cham.Edur 1991.68.10-12. sivasuhramanlam. S.;Talihudeen, 0 . J. Sci. Food Agr. 1971.22.325-329. van Faber, F.C.Flom 1925.118,89. See e g . , Huheey. J. E. lnorponir Chamistry, Pn'neiplel 01 Stlucture and Reocfiuily. 3rd ed.; Harper and Row: 1983: Chsgfer 6. 8. See.e.e.Levine. B.A.: Wil1isms.R. J.P.inColeivmondCellFuncIion;Choung,W.Y., 1.
2. 3. 4. 5. 6. 7.
"p
12. ~ e u e lH.; , whartman, J.: Hu~ehneker,K.;sehohinger,u . ; ~ u d e l , C ndu. . Chim.~cto 1959, 42, 1160-1165: Halasz, I.; Sebastian, I. Angew. C h m Infernal., E n d Ed. 1969.8.453454. 13. Alherk, B.; Bray, D.: Lewi8.J.; Rsff. M.: Roberts, K.; Watnan. J. D. Molerular Biology olrhr Cell, 2nd ed.; Garland: 1989:Chapter 14. 14. Hauser,H.;Levine,B. A ; Williams, R.J. P, T~endaBiochem.Sci. 1976,1,27%281. 15. See,e.g.,Trig610,D. J. Pm,e.Sur/ace Mombmnr Sci. 1972,6,267-331. 16. E V ~ I SD; ~ ,O'connor, M., Eds. Silicon Biochemistry; Wiley: 1986. 17. Sehwsrtz,K.Proe.Nol. Acod Sci. 1 9 7 3 , 7 0 , 1 6 0 ~ 1 6 1 2 . IS. S~hwarrr,K. In Biochemistry o/Siliron ond Ralotzd Problems: Bendz, G.: Lindqvist, I. Eds.: Plenum: 1978: pp 207-230; Sehwsrtr. K. Lancet 1977.454-456.
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19. Carlidc, E. M. In Silicon Biochemistry; Evered, D.:O'Connor, M., Ed..: Wiloy: 1986: pp 123-136. 20. Riedel, W. R.: Senfilippo, A. In Silicon and Siliceous Structures in Biologieol Sysfrma: Simpaon, T. L.:Voleani, B. E.. Ed% Springer: 1981: pp 322-346. 21. sangstor, A. G.;Hodson, M. J. I" Silion Biochemisfry; Evered. D: O'connor, M., Eds; Wiley: 1986:pp 90-103. 22. Ksufman. P.B.: Dsyanandan,P.:Takeoka,Y.;Bigelaw,W. C.; Jones,J. D.: 1ler.R. K. In Silicon and Siliceous Structures in BiologicolSystema: Simpson, T. L.; Voleeni, B. E.. Ed% Springer: 1981: pp409-449. 23. For recent reviews see: Lowenstoin, H. A,; Weiner. S. On Minemiirafion; Oxford University: 1989: Simkiss, K.: Wilbur, K. M. Biomineroliration. Cell Biology ond Minemi Deposits; Academic: 1989. 24. Heeky,R.E.:Mopper, K.:Kilman,P.: Degens,E.T. MorineBiol. 1973,19,328-331. 25. Volcani, B. E. In Silicon and Siliceous Structure8 in Biologicel Systems; Simpsan, T. L..Volcani, B. E.. Ed..: Springer: 1981: pp 157-200. 26. Sullivan, C. W. In Silieon Biochemistry; Evered, D.; O'Connor, M., Eda.: Wiley: 1986: pp 59-86.
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32. Kiachvink,J. L.: Jones, D. S.: MaeFsdden, B. J., Eds. ~ ~ Minerdimtion & i ~ and Mametoroeoption in Organisms; Plenum: 1985. 33. Candy. J. M.: Oakl~y,A.E.:Klinowski.J.: Carponter,T. A.:Perry.R. H.:Atack,J.R.: P m y , E. K.: Blessed. G.; Fairbairn, A. E.; Edwardaan, J . A. Loncar 1986, 354: Candy. S. M. In Silicon Edwardson. J. A ; Klinowski, J.: Oakley, A. E.; Perry. R. H.; Biochemiafry; Evered, D.; O'Cannor, M., Ed..; Wiley: 1986:pp 160.173, 34. Birchall, J. D. ChemB~ifninI990,26,141-144.
