Inhibition of Calcium Carbonate and Phosphate ... - ACS Publications

Chapter 5

Inhibition of Calcium Carbonate and Phosphate Crystallization by Peptides Enriched in Aspartic Acid and Phosphoserine 1

Downloaded by UNIV OF ARIZONA on August 1, 2012 | http://pubs.acs.org Publication Date: November 26, 1991 | doi: 10.1021/bk-1991-0444.ch005

C. Steven Sikes, M. L. Yeung, and A. P. Wheeler

Mineralization Center, University of South Alabama, Mobile, AL 36688 Polyanionic peptide analogs of matrix proteins from biominerals were synthesized by automated, solid-phase methods. The peptides were used to evaluate the chemical requirements for inhibition of calcium carbonate and phosphate crystallization as measured using pH-drift and constant-composition assays. Continuous runs of negatively charged residues were required for maximum inhibitory activity. Asp was the optimum size for inhibition of CaCO crystal growth, but Asp exhibited maximal inhibition of CaCO crystal nucleation. A n hydrophobic domain of Alas added to Asp led to increased inhibition of CaCO crystal nucleation but had no effect on crystal growth. Adding the hydrophobic domain to Asp did not enhance inhibition of either CaCO nucleation or crystal growth. The results suggest that crystal nucleation can be suppressed through diffusion-limitation related to the presence of a barrier at the crystal/solution interface. Asp molecules seemed to fill both the crystal-binding sites and the zone of diffusion around them, with additional anionic or hydrophobic residues simply contributing excess mass without enhancing performance. CaCO crystal growth appeared not limited by diffusion but rather by some other process such as incorporation of already bound ions into the lattice. Phosphorylation of N terminal serine residues by use of monochlorophosphoric acid produced H-PSer -Asp -OH, the most powerful inhibitor of both calcium carbonate and calcium phosphate formation yet discovered. This supports the importance of phosphorylated residues to the function of protein inhibitors of mineralization 15

3

(30 to 40)

3

15

3

40

3

40

3

(1

to

3)

20

1

Current address: Department of Biological Sciences, Clemson University, Clemson, SC 29634-1903 0097-6156/91/Q444-0050$06.50/0 © 1991 American Chemical Society In Surface Reactive Peptides and Polymers; Sikes, C., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

5. SIKES ET AL. Inhibition of Crystallization

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and suggests that the terminal residues may play a significant role in the mechanism of inhibition. Proteins that inhibit calcium carbonate and phosphate formation in vitro occur as part of the organic matrix of biominerals and as components of saliva (1). These proteins are thought to function in part to stop or start crystal formation as needed in the case of matrix, depending on whether they are in solution or immobilized (2-4), or to stabilize enamel and saliva in the case of the salivary proteins (5). The attempt to understand how these molecules interact with crystal surfaces is aided by consideration of their common structural features. For example, recent evidence suggests that several matrix inhibitors of crystallization may have polyaspartic acid domains (6-11). Moreover, virtually all soluble matrices are at least enriched in negatively-charged aspartic and glutamic acids (1,12). One hypothesis holds that aspartic acid residues may alternate with small, neutral amino acids in matrix (13). Consequently, it is of interest to determine whether polyaspartic acid domains are required for inhibition of crystallization by matrix and to what extent the length of the domain influences this activity. Phosphoserine is also a prominent component of many matrix molecules (1415), and in some cases there may be continuous runs of this residue as well (16-17). The importance of phosphoserine to activity of proteins as regulators of crystallization has clearly been seen in studies of the salivary proteins. In the case of the proline-rich phosphoproteins (PRP) from saliva (18-19), removal of phosphate groups by treatment with alkaline phosphatase greatly diminished the activity of the molecules. Similar observations had been made for phosvitin, the phosphoprotein from egg yolk (20), and more recently for proteins from oyster shell (1). Therefore, another point of interest is the extent to which phosphoserine is needed for regulation of crystallization by proteins that may already be polyanionic, especially those including runs of aspartic residues. The possible importance of the placement of phosphoserine residues is also unknown. For example, in some proteins like the PRP's (21), phosphoserine may be dispersed, but in statherin (22), the two phosphoserine residues are at the N-tenriinus. Accordingly, the effect of having phosphoserine residues at specific locations on the ability of a protein to regulate crystallization is another topic for study. Finally, the polyanionic protein inhibitors of crystallization invariably also contain hydrophobic domains that may be quite extensive. One possible function, among several suggested functions, of these domains has been formulated from the effects of statherin on calcium hydroxyapatite formation (23). The hydrophobic portion of statherin, consisting in the bulk of the 43-residue molecule, was required for maximum inhibition of crystallization. It seemed possible that statherin bound to crystal surfaces via the polyanionic N-terminus, blocking crystal growth there, while the rest of the molecule extended from the surface of the crystals, disrupting movement of lattice ions to the surface.

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


52

SURFACE REACTIVE PEPTIDES AND POLYMERS

This concept has been tested in part by studying the effects of polyaspartate molecules with and without hydrophobic domains of polyalanine on CaCC>3 crystallization (24). The results suggested that the presence of a hydrophobic region could improve the inhibitory activity of polyaspartic-containing molecules. However, it was not known exactly what relative lengths of polyanionic and hydrophobic domains were required for effective interaction with forming crystals. In view of these observations, the present study was undertaken 1) to determine if polyaspartic domains optimize inhibitory activity of proteins with respect to crystallization, 2) to determine the size of polyaspartic domains that interact most effectively with crystal nuclei and preformed crystal surfaces, 3) to demonstrate the influence of phosphoserine residues on inhibition of crystallization by proteins, including the possible effect of the location of phosphoserine residues, and 4) to further evaluate the possible influence of hydrophobic domains on crystallization inhibitory activity by peptides of different sizes. The general approach was to synthesize model peptides and to measure effects on crystal nucleation and crystal growth of calcium carbonate and calcium hydroxyapatite. Materials and Methods Preparation of Peptides. An automated, solid-phase peptide synthesizer (Applied Biosystems, Model 430A) was used to prepare peptides. Aspartic acid was supplied as t-Boc-L-aspartic acid with beta carboxyl protection by Obenzyl linkage, serine as t-Boc-L-serine-O-benzyl, alanine as t-Boc-I^-alanine, and glycine as t-Boc-I^ glycine (all from Applied Biosystems). Standard reaction cycles were used. In all cases, the coupling efficiency of each residue was checked by automated sampling of peptide resin for measurement of unreacted free amine by the ninhydrin method (25). Coupling efficiencies routinely were greater than 99% per cycle of synthesis. Cleavage. Following synthesis, peptide-resin was repeatedly washed with methanol, then air-dried and weighed. Next, peptides were cleaved from the resin using a modification of the trifluoromethyl sulfonic acid (TFMSA) procedure at -10 °C as a precaution to suppress aspartimide formation (26), in which the beta carboxyl group of aspartic acid undergoes a dehydration reaction with the secondary amine of an adjacent residue along the peptide backbone to form a 5-membered ring (27). For 100 mg samples, peptide-resin in a scintillation vial was treated for 10 minutes with 150 ml of anisole to swell the resin, making it more accessible for reaction. Then 1.0 ml of neat trifluoroacetic (TFA) was added with magnetic stirring and allowed to react for 10 minutes. Next, 100 μΐ of concentrated TFMSA (Aldrich Chemical Co.) were added with cooling using a salt-water ice bath at -10 °C with cleavage for 30 minutes, followed by 30 minutes at room temperature. For cleavage of other amounts of peptideresin, the amounts of the reagents were changed proportionally.


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SIKES ET AL.

Inhibition of Crystallization

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Washing. Following cleavage, 20 ml of methyl butyl ether (MBE, Aldrich) were added to the vial to ensure precipitation of the peptide, which already was relatively insoluble in the acidic reaction medium because of the acidic nature of the peptides. After stirring for 1-2 minutes, the entire slurry was passed through a 4.25 cm glass fiber filter (Fisher G4) using a filter funnel and vacuum at 15 psi. This began removal of TFA, TFMSA, anisole, and any soluble reaction products, leaving the cleaved peptide and resin on the filter. The peptide on the filter was washed several times with a total volume of 100 ml of MBE. Extraction. The insoluble acid form of the peptide on the filter was converted into a soluble sodium salt by extraction into a clean, dry flask with 10 ml of Na2CC>3 (0.02 M , pH 10.2), using 5 successive rinses of 2 ml, with at least 1 minute extraction on the filter prior to applying the vacuum each time. This produced afiltrateof about pH 5 that contained the solubilized peptide. Purification. The filtrate was dialyzed twice with stirring against 2 liters of distilled water for 2 hours each time using dialysis tubing (Spectrapor, nominal M W cutoff of 1000 daltons). The dialysate was frozen and lyophilized, yielding white flakes or powders. The average yield of peptides was 40%. Purity of peptides was checked by high performance liquid chromatography (Varian 5500 L Q using gel permeation columns designed for peptides (Toya Soda 2000 SW and 3000 PWXL). Single, sharp peaks at the appropriate MW's were obtained (24). Both anion-exchange and reverse-phase liquid chromatography of the peptides also produced well-defined, single peaks. The anion-exchange column (Synchrome, Inc., Synchropak AX300) was run with a continuous gradient of distilled water to 2.0 M NaCl in 1 hour at 25 °C. The reverse-phase column (Phenomenex, ultracarb 5 0DS 30) was run with a continuous gradient of 0.05 M sodium acetate (pH 6.5) to 100% acetonitrile in 20 minutes at 25 °C. Because there were no peaks in the hydrophobic portion of the gradient, few if any peptides having uncleaved benzyl protecting groups were present. Phosphorylation of Peptides. Monochlorophosphoric acid (H2PO3CI), produced by reacting one equivalent of phosphorus oxychloride (POCI3, Aldrich), with 2 equivalents of water, was used as the phosphorylating agent (28-29). Because this reagent has only one reactive site, it is not as prone to inappropriate reactions as may occur with use of POCI3 (30). Numerous attempts to phosphorylate polyanionic peptides by use of diphenylchlorophosphate (Aldrich) were unsuccessful due to irreversible binding of the peptides to the catalysts during the hydrogénation step to remove diphenyl protecting groups. In addition, there was evidence that some cleavage of the polyanionic peptides occurred with use of this reagent. Although diphenylchlorophosphate has been used successfully in synthesis of phosphopeptides having one or two serine residues (31-32), its use appears to lead to problems with larger, more polyanionic peptides.



54


An example protocol follows. Phosphorus oxychloride (1.872 ml, 20 mmole) was added over a period of one hour to water (0.72 ml, 40 mmole), followed by stirring for 30 minutes to allow completion of formation of monochlorophosphoric acid. Next, approximately 100 mg of peptide (in the case of Asp2oSer2, for example, this represented about 0.0% mmoles of serine residues) were added, followed by stirring at 60 °C for 4 hours. The reaction was ended by dropwise addition of 0.36 ml (20 mmole) of water to degrade any unreacted monochlorophosphoric acid to orthophosphate. Next, IN HC1 (approximately 1.3 ml) was added to give a solution of 0.3 N HC1. This was heated in a boiling water bath for at least 20 minutes to destroy any polyphosphates that may have formed. Upon cooling, 10 ml of MBE was added to precipitate the phosphopeptide, followed by centrifugation and multiple washings with 5 ml of methanol. After lyophilization, the peptide was dissolved in distilled water, adjusted to about pH 7 with IN NaOH. This solution was dialyzed twice against 2 liters of distilled water for 4 hours. The dialysate was lyophilized to give the final product. The extent of phosphorylation of peptides was measured spectrophotometrically upon formation of the phosphomolybdate complex (33). All phosphate present was bound, requiring release by digestion in persulfate-H2SC>4: there was no soluble reactive phosphate in the purified peptide samples. Verification of Peptide Structures, a) Peptide Sequencing. An automated protein sequencer (Applied Biosystems, model 477A with an on-line amino acid analyzer, ABI model 120A) was used to confirm the primary structures of the peptides. Standard cycles based on Edman degradations were used. Amino acids were identified as their phenylthiohydantoin derivatives using PTH standards (ABI). b) Alkalimetric Titrations. Solutions of peptides (5 ml at 1 mg/ml) were titrated with 0.1 Ν NaOH over pH 2 to 12. The amount of base required to neutralize the carboxyl groups (pKa approx. 4.0) and phosphate groups (pKa=7.10) was used to calculate the amounts of these groups present (34). c) ^-elimination of phosphate. Phosphate O-linked to serine is removable by a base-catalyzed B-elimination reaction in which there is an increase in absorbance at 240 nm due to the production of dehydroalanine as a reaction product (3536). Samples of peptides at 0.1 mg/ml in 0.5 Ν NaOH at 24°C were monitored in a quartz cuvette continuously at 240 nm (Perkin Elmer UV/VIS, model lambda 4A).

Crystallization Assays, a) CaC03 Screening Assay. An assay fashioned after Hay et al. (5) was used to allow comparison of general activity of multiple inhibitors in one set of measurements. Solutions supersaturated with respect to CaCOe were prepared by separately pipetting 0.2 ml of 1.0 M CaCfe dihydrate and 0.4 ml of 0.4 M NaHCOs into 19.4 ml of artificial seawater (0.5 M NaCl, 0.011 M KC1). This resulted in initial concentrations of 10 mM C a and 8.0 m M dissolved inorganic carbon (DIC). Actual concentrations of C a were confirmed 2+

2+



5. SIKES ET AL.


55

by atomic absorption and of DIC by Gran titration (37). The reaction vessels were 30 ml bottles with screwtops. These were partially immersed in a 1-gallon, plastic aquarium fitted for flow-through to a thermostated recirculating water bath (VWR model 1115) at 20 °C containing a submersible magnetic stirplate with 15 stirring locations (Cole Parmer). Crystallization was initiated by adjusting pH upward to 8.3 by titration of μΐ amounts of 1 Ν NaOH. Final pH was read after 24 hours. Following addition of Ca but before addition of DIC, peptides were added to reaction vessels from stock solutions of 1.0 or 0.1 mg peptide/ ml. After experiments, reaction glassware was washed with 0.1 N HC1 for at least 10 minutes followed by a treatment of at least 10 minutes with a solution of sodium hypochlorite (5.25 /ig NaOCl/ml, a 1/1000 dilution of commercial bleach). These treatments were necessary to dissolve crystals that formed on the surface of the glassware and to destroy any peptides that may adhere to the glass. Failure to perform either treatment led to abnormal results in control experiments. Following HC1 and NaOCl treatments, reaction glassware was rinsed with several volumes of distilled water and dried for use in subsequent experiments. Results in control experiments demonstrated the importance of using the same reaction vessels for a set of measurements. b) CaC03 Nucleation. The reaction conditions were the same as above. The reaction vessel was a 50 ml, 2-necked, round-bottom flask partially immersed in a thermostated water bath at 20 °C. The reaction vessel was closed to the atmosphere to minimize exchange of CO2. The reaction was started by adjusting the pH upward to 8.3 by titration of μΐ amounts of 1 Ν NaOH by digital pipette. The reaction was monitored by pH electrode and recorded by strip chart. In these assays, there is first an induction period of stable pH of about 6 minutes during which crystal nuclei form, followed by a period of crystal growth when pH drifts downward due to removal of carbonate from solution. Inhibition of nucleation is indicated by the length of the induction period. c) Seeded CaŒ>3 crystal growth. The reaction conditions were modeled after Kazmierczak et al. (38) and consisted in 50 ml of artificial seawater as above with 2 m M each of CaCfe and DIC at pH 8.5. The reaction vessel was a 100 ml, water-jacketed, glass cylinder with a stoppered topfittedwith a pH electrode and 2 burette delivery tips. The reaction was thermostated at 20 °C. The solution was stable until initiation of the reaction by addition of 2.5 mg of CaŒ>3 seeds (Baker Analytical) which began to grow immediately. Seeds were aged prior to use by stirring as a slurry of 100 g CaC03 in 1 liter of distilled water for 3 weeks in a closed vessel. This aging of seeds was necessary to provide reproducible control curves. In some cases, 1 ml batches of aged seeds were sealed in ampules until opening for use. It also was acceptable to pipette seeds directly from the 1 liter slurry. However, frequent opening of the slurry to the atmosphere may lead to changes in the solution with resultant changes in the seeds, probably due to exchange of atmospheric CO2. Peptides were added to the reaction vessel prior to the addition of the seeds. Reaction conditions during growth of seeds were held constant by autotitration of stocks of 0.1 M each of CaCk and Na2CX>3


56


(pH 11.1) from separate burettes controlled by a computer-assisted titrimeter (Fisher CAT). This replenished the lattice ions to the solution as they were removed due to crystallization and kept the pH at 8.50±0.02. d) Calcium phosphate crystallization. An assay modified after Termine and Conn (20) was used. A solution supersaturated with respect to calcium phosphate was prepared by separately pipetting 0.1 ml of 1.32 M CaCb dihydrate and 0.1 ml of 0.90 M N a H P 0 into 29.8 ml of distilled water. This yielded initial concentrations of 4.4 mM C a and 3.0 mM dissolved inorganic phosphorus (DIP). The reaction vessel was a 50 ml, round-bottom, 2-necked flask partially immersed in a thermostated water bath at 20 °C. The reaction vessel was closed to the atmosphere. The reaction began upon mixing the reactants with an initial pH of 7.4. In experiments, peptides were added after the calcium and before the DIP. Amorphous calcium phosphate (ACP) nucleates immediately upon addition of DIP and slowly grows as indicated by a modest decrease in pH during the first 30 minutes or so of the assay. Following this, A C P begins to transform to calcium hydroxyapatite (HAP), Caio(P04)e(OH)2, as indicated by a marked acceleration in the downward pH drift. The reaction ceases as reactants are depleted and the pH is lowered. 2

4


2+

Results Polyaspartate and Polyaspartate-alanine Molecules. The enhancement of inhibition of CaCU3 nucleation associated with an hydrophobic domain of alanine residues attached to a polyaspartate molecule is shown in Figure 1. An effect on crystal nucleation was clearly seen as an increase in the induction period prior to crystal nucleation as a function of length of hydrophobic domain. In the case of AspisAlae, addition of the hydrophobic domain was even more effective in these assays than increasing the number of Asp residues to 25. In keeping with routine practice in polymer science, as pointed out in a prior study (24), results herein are reported on a weight basis rather than a molar basis because several smaller molecules of a homogeneous polymer may be functionally equivalent to one larger molecule of the same polymer. Therefore, the total mass of polymer rather than the total number of molecules can be the functionally important parameter. A possible effect of the polyaspartate-alanine molecules on crystal growth was obscured in pH-drift measurements due to the changes that occur in pH and concentrations of lattice ions during crystal growth in the assays. Therefore, a constant composition, seeded-crystal approach to demonstrate effects on crystal growth was taken. An example experiment is shown in Figure 2 in which a control rate of crystallization was established, followed by sequential addition of increasing doses of a polyaspartate until crystallization ceased. This type of experiment was run for a set of molecules including polyaspartatds alanine^ to ι ο and polyaspartaten 5 and 25) (Figure 3). In contrast to thefindingsfor nucleation,



5. SIKES ET A L

Control


Aspi

5

Aspi Ala 5

2

Aspi Ala 5

4

Aspi Ala 5

5

Aspi Ala 5

57

6

Aspi Ala 5

8

Asp 5 2

P E P T I D E S (0.05/ig/ml)

Figure 1. The effects of Aspis, and Asp25, and Aspi5Ala on CaCC>3 nucleation. Means ± standard deviations, n=3 to 9.


58


100


I

Aspis

Asp25

Aspi Ala 5

2

Aspi Ala 5

4

Aspis A l a

6

Aspis A l a

8

A s p i Alaio 5

P E P T I D E S (0.1 /ig/ml)

Figure 3. The effects of Aspis, Asp25, and AspisAla^toio) on growth of CaCU3 seed crystals. Means ± standard deviations, n=3.



5.

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59

there was no enhancement of inhibition of crystal growth related to the presence of an alanine domain, indicating that inhibition of crystallization by these peptides was due only to an effect on nucleation. The general inhibitory effect of a family of polyaspartate molecules on CaCOe formation as measured by use of the screening assay is shown in Figure 4. At a molecular size of about 30 to 40 residues, polyaspartate was most effective as an inhibitor as shown by the lowest dose (0.15 mg/ml for Asp3o and 4o) that stabilized the pH of the solutions for 24 hours. To confirm these results and to distinguish between effects on crystal nucleation and crystal growth, both nucleation assays (Figure 5) and crystal growth assays (Figure 6) were run using these peptides. Again, the most active polyaspartate versus crystal nucleation was in the range of 35 residues. Surprisingly, however, the most active versus crystal growth was Aspi 5. Although Aspi 0 had moderate activity (24), Asps was ineffective as an inhibitor of CaC03 crystallization (data not shown). Knowing that Asp4o was a particularly effective size of polyaspartate for general inhibition of crystallization, it was of interest to determine the effect of adding a polyalanine domain to this longer polyanion, especially for comparison to the Aspis series. As seen in Figure 7, Asp4o still performed better than Asp4oAlae in the CaCOe screening assay, although the Asp4oAlae seemed to perform better than Asp45 molecules. A lack of effect on crystallization resulting from addition of the alanine domain to Asp4o was confirmed by use of both the CaCOe nucleation assay (Figure 8) and the crystal growth assay (Figure 9) in that neither showed enhancement of inhibition imparted by the alanine domain. Phosphoserine-polyaspartate molecules. The addition of one to three phosphoserine residues onto Asp2o molecules resulted in a remarkable enhancement of both CaC03 (Figure 10) and calcium phosphate crystallization (Figure 11) relative to polyaspartate molecules of appropriate sizes. The effect on calcium phosphate formation indicated a surprisingly broad activity for these molecules in that although polyaspartate is a strong inhibitor of CaCU3 formation, it is not particularly effective versus calcium phosphate formation. The effect of the PSer-containing Asp2o molecules on calcium phosphate crystallization apparently was mainly at the level of conversion to apatite since amorphous calcium phosphate still formed early in the experiments and grew steadily but the sharp breaks in the curves indicative of apatite formation did not occur (Figure 11). A more detailed analysis of the performance of the PSer-containing Asp2o molecules is given in Table 1. Notice that both the Asp2oPSer, and Asp2oPSer2 molecules were essentially completely phosphorylated based on the analysis of % phosphate by weight of the peptides. However, for reasons that are not clear, the Asp2oPSer3 molecules on the average had only about 2 out of 3 serine residues phosphorylated.


60



Control

Aspio

Asp o 2

Asp3o

Asp3

5

Asp4o

Asp o 5

Aspeo

P E P T I D E S (0.05Afg/ml)

Figure 5. The effects of polyaspartate molecules of different sizes on CaC03 nucleation. Means ± standard deviations, n=4 to 10.


5.

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61

100

I

ο

ce Ο _ι < Ico

>

oc


ο

LU CO < LU OC Ο LU Q Asp o

Asp o

3

Asp
3 nuclei or amorphous calcium phosphate. Indeed several of the protein inhibitors of mineralization are known to have polyanionic terminal regions (1,6,7,11), and statherin in particular has an N-terminus consisting in H-AspPSer-PSer-Glu-Glu- (22). In some cases, there is evidence that polyelectrolytic polymers do bind to crystal at one end, with the rest of the molecule extending from the surface (56). The presence of uncharged spacer residues, such as glycine or serine, between or interspersed among anionic residues of peptides led to considerably reduced inhibitory activity in all of the assays. Although phosphorylation of serine residues improved this activity in all cases, optimum activity of protein crystallization inhibitors still seemed to require continuous runs of anionic amino acids. As discussed elsewhere (1,7,8,24), although it is possible that ordered copolymers of (Asp-X) and (Asp-X-Y) , where X and Y are small, neutral residues (12,13,57), exist to some extent in matrix proteins, such sequences do not account for the observed activity of the proteins as crystallization inhibitors. Finally, the study of biomineralization inhibitors has produced a number of molecules that have uses ranging from biomedical to industrial applications (58,59). For example, Williams and Sallis (60) established that phosphocitrate was the most potent inhibitor on a molar basis of hydroxyapatite formation among a group of materials including 1-hydroxyethane 1,1-diphosphonate (EHDP), perhaps the most widely commercialized organic crystallization inhibitor. As seen in the present studies, the polyanionic peptides with phosphoserine N-termini (MW 2500) although not as potent as phosphocitrate (MW 272 g) on a weight basis, are the most potent inhibitors of calcium carbonate and phosphate crystallization on a molar basis yet discovered. The design of this study required analysis of the effects of the peptides on a weight basis. The analysis of molar binding characteristics, molar affinities, surface coverage of crystals, and inhibition of CaCOe crystallization by selected polyanionic peptides is the subject of a following paper (61). n

n

Acknowledgments This work was supported in part by grants from the Alabama Research Institute, the Mississippi-Alabama Sea Grant Consortium, the National Science Foundation,



69

and the Office of Naval Research. We thank J. D. Sallis for providing a sample of phosphocitrate.


Literature Cited 1. Wheeler, A. P.; Sikes. C. S. In Biomineralization: Chemical and Biochemical Perspectives; Mann, S.; Webb, J.; Williams, R. J. P., Eds.; V C H Publishers: Weinheim, W. Germany, 1989, p 95. 2. Crenshaw, Μ. Α.; Linde, Α.; Lussi, A. In Atomic and Molecular Processing of Electronic and Ceramic Materials; Aksay, I. Α.; McVay, G. L.; Stoebe, T. G.; Wager, J. F., Eds.; Materials Research Society: Pittsburgh, 1988; p 99. 3. Linde, Α.; Lussi, Α.; Crenshaw, M . A. Calcif. Tissue Int. 1989, 44, 28695. 4. Gunthorpe, M . E.; Sikes, C. S.; Wheeler, A. P. Biol. Bull. 1990, in press. 5. Hay, D. I.; Schluckebier, S. K.; Moreno, E. C. Calcif. Tissue Int. 1986, 39, 151-60. 6. Butler, W. T.; Bhown, M . ; Dimuzio, M . T.; Cothran, W. C.; Linde, A. Arch. Biochem. Biophys. 1983, 225, 178-86. 7. Rusenko, K. W. Ph.D. Dissertation, Clemson University, Clemson, South Carolina, 1988. 8. Wheeler, A. P.; Rusenko, K. W.; Sikes, C. S. In Chemical Aspects of Regulation of Mineralization; Sikes, C. S.; Wheeler, A. P., Eds.; University of South Alabama Publication Services: Mobile, AL, 1988; p 9. 9. Wheeler, A. P.; Low, K. C.; Sikes, C. S. In Surface Reactive Peptides and Polymers: Discovery and Commercialization; Sikes, C. S.; Wheeler, A. P., Eds.; ACS Books: Washington, D. C., 1990. 10. Gorski, J. P.; Shimizu, K. J. Biol. Chem. 1988, 263, 15938-45. 11. Robbins, L. L.; 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. 12. Weiner, S. Crit. Rev. Biochem. 1986, 20, 365-408. 13. Weiner, S. Biochemistry 1983, 22, 413945. 14. Veis, A. In The Chemistry and Biology of Mineralized Tissues; Butler, W. T., Ed.; Ebsco Media; Birmingham, AL, 1985; p 170. 15. Veis, Α.; Sabsay, B.; Wu, C. Β. In Surface Reactive Peptides and Polymers: Discovery and Commercialization; Sikes, C. S.; Wheeler, A. P., Eds.; ACS Books: Washington, D.C., 1990. 16. Williams, J.; Sanger, F. Biochem. Biophys. Acta 1959, 33, 294- 6. 17. Lechner, J. H.; Veis, Α.; Sabsay, B. In The Chemistry and Biological of Mineralized Connective Tissues; Veis, Α., Ed.; Elsevier North Holand: Amsterdam, 1981, p 395. 18. Aoba, T.; Moreno, E. C.; Hay, D. I. Calcif. Tissue Int. 1984, 36, 651-8.



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