American Mineralogist, Volume 82, pages 878±887, 1997

Surface site-speci®c interactions of aspartate with calcite during dissolution: Implications for biomineralization

H. HENRY TENG AND PATRICIA M. DOVE School of Earth and Atmospheric Sciences, Georgia Institute of Technology, Atlanta, Georgia 30332-0340, U.S.A.

ABSTRACT Calcite occurs widely as a component in the exoskeletons and tissues of marine and freshwater invertebrates. Matrix macromolecules involved in regulating the biological growth of calcite in these organisms are known to share a carboxylic-rich character that arises from an abundance of the acidic amino acids aspartate (Asp) and glutamate (Glu). This study determines the interactions of Asp with calcite {1014} faces during dissolution using in situ ¯uid-cell atomic force microscopy (AFM) and macroscopic ex situ optical methods. In control experiments, etch-pit morphologies produced by dissolution in simple undersaturated solutions re¯ect the inherent symmetry of the {1014} faces with a rhombus form. With the introduction of Asp, surface site reactivities are modi®ed to yield isosceles triangular etch pits and hillocks. With continued exposure to Asp-bearing solutions, these triangular pits coalesce and the surface evolves into a network of interconnected tetrahedral etch hillocks. The component tetrahedral ``sides'' have Miller-Bravais indices of (0001), (1101), and (0111). These faces intersect the (1014) face in the [010], [451], and [411] directions to compose the three edges of the triangular etch pits. Structural and stereo- chemical contraints suggest that the (1101) and (0111) faces in the hillock are a combi- nation of corresponding faces from the {1102} and {1100} crystallographic forms. Results of this dissolution study are consistent with previous growth experiments show- ing that Asp causes preferential development of the {0001} and possibly the {1100} forms of calcite. These observations support mechanisms proposing that the new forms are sta- bilized by the molecular recognition of Asp functional groups for speci®c surface sites. Because Asp stabilizes identical faces during growth and dissolution, we suggest that dissolution studies offer an alternative means of determining the crystal forms that develop during biomineralizing processes and a more direct means of identifying those surface sites involved. We demonstrate that the stability of crystallographic directions expressed by step edges is controlled by the relative reactivities of surface sites. Our ®ndings yield new insights into surface structure controls on mineral reactivity.

INTRODUCTION mineral precipitation. These lack known biolog- Biomineralization is the process by which the biolog- ical function (Skinner 1993). In this type of bio- ically mediated activities of organisms to mineral mineralization, biological systems exercise little direct nucleation and growth. This process has been divided into control over mineral formation although biological sur- two fundamentally different types based on the degree of faces may be important in the induction of processes. At biological control (Lowenstam 1981). In biologically con- least 60 carbonate, phosphate, oxide, sulfate, and sul®de trolled mineralization, morphologically complex struc- minerals can be formed through biomineralizing activities tures nucleate and grow in concert with a genetically (Simkiss and Wilbur 1989). Of these, the calcium car- programmed macromolecular matrix of proteins, poly- bonates, calcite and aragonite, have the most widespread saccharides, and lipids. The resulting mineral microar- occurrence and form through either type of chitectures ful®ll speci®c physiological functions such as biomineralization. the addition of stiffness and strength to skeletal tissues (e.g., Berman et al. 1993; Mann 1993). These ``bioma- Previous studies of organic-calcite interactions terials'' can also have remarkable physical properties that Several studies have investigated processes governing cannot be imitated by the most sophisticated synthetic biomineralization by examining calcite growth morphol- materials (Vincent 1990). In contrast, biologically in- ogies produced in the presence of proteins, amino acids, duced mineralization occurs as a result of interactions be- and other organic compounds. The matrix macromole- tween the biological activities of an organism and its sur- cules involved in regulating biological growth of carbon- rounding physical environment to produce secondary ate minerals share an acidic character created by an abun- 0003±004X/97/0910±0878$05.00 878 TENG AND DOVE: ASPARTATE INTERACTION WITH CALCITE 879 dance of the amino acids aspartate (Asp) and glutamate servations of nanoscale reaction processes. Fluid-cell (Glu) (Crenshaw 1972; Weiner et al. 1983; Weiner 1986; AFM records the evolution of mineral surface microto- Addadi et al. 1987; Lowenstam and Weiner 1989). In pography, individual step heights, and relative rates of vitro studies suggest that these macromolecules modify step migration as they occur in aqueous solutions. Pre- calcite morphology by selectively binding to certain crys- vious studies demonstrate the utility of AFM for under- tal faces and inhibiting growth (Addadi and Weiner 1985; standing calcite surface structures and reactivity. AFM Addadi et al. 1987; Berman et al. 1988; Mann et al. 1990; has been used to investigate calcite growth and dissolu- Berman et al. 1995). When adsorbed onto a solid tion processes, to observe the formation or retreat of the substrate, they induce the formation of unusual crystal- mono-molecular layer 3 AÊ steps on the (1014) lographic faces such as (0001). For example, Addadi and surfaces (Hillner et al. 1992a, 1992b; Gratz et al. 1993; Weiner (1985) and Addadi et al. (1987) demonstrated that Dove and Hochella 1993; Stipp et al. 1994) and to study Asp-rich proteins and ␤-sheet polyaspartate adsorbed on the con®guration of molecular-scale surface structures sulfonated polystyrene surfaces lead to the formation of (Ohnesorge and Binnig 1993; Stipp et al. 1994). This pa- calcite (0001) growth faces. Berman et al. (1988) ob- per continues the investigation of controls on carbonate served that the acidic macromolecules extracted from sea surface reactivity by examining the in¯uence of Asp on urchin tests stabilized prismatic faces. Mann et al. (1990) calcite microtopography. found that ␣-aminosuccinate (aspartate) and ␥-carboxy- Aspartate-calcite interactions are the focus of our study glutamate were more ef®cient at stabilizing (promoting because of the documented importance of this system to the formation of ) ®rst-order prismatic {1100} faces than biomineralization. Previous studies report the in¯uence of second-order prismatic {1100} growth faces. They also Asp on calcite growth using ex situ macroscopic ap- showed that amino-functionalized carboxylates (amino proaches, but none has determined the microscopic con- acids) are more effective than their simple carboxylic acid trols of this amino acid on growth or dissolution by in counterparts at stabilizing the formation of these new fac- situ microscopic methods. Recognizing that experiments es. These observations suggest that acidic amino acids using the monomeric form of aspartic acid do not dupli- in¯uence growth through the orientation and interaction cate the Asp residue found in natural proteins, we inves- of their side-chain carboxylates with calcite surfaces and tigated the crystallographic-speci®c interactions of Asp their ability to create extended Ca-interacting domains. with calcite surfaces with the goal of answering the fol- Thus, surface-speci®c interactions of growth modi®ers lowing questions: (1) How does Asp modify the surface are believed to be an essential controlling factor in de- topography of calcite during dissolution? (2) Is it possible velopment of diverse morphologies for the minerals in to identify the site-speci®c interactions of organic growth biological systems. modi®ers through studies of dissolution? (3) Do similar These investigations of mineral formation in solutions surface-speci®c interactions occur during growth and dis- containing diverse organic constituents remind us that we solution to stabilize development of the same crystal fac- lack a consistent mechanistic model that can describe and es? Answers to these questions are a ®rst step in our predict the interactions of organic compounds with min- long-term goal of characterizing the controls of complex eralizing surfaces. This continuing gap in our scienti®c organic compounds on mineral formation in knowledge also hinders our ability to predict or control biomineralization. carbonate precipitation and growth in other natural or en- The use of dissolution studies as a tool for determining gineered systems where organic compounds are present. crystal symmetry is well documented in mineralogy and However, the literature contains clues suggesting that a crystallography (e.g., Zoltai and Stout 1984; Putnis 1992) consistent framework for a mechanistic model exists. Pre- and materials science (e.g., Sangwal 1987). In a similar vious biomineralization studies suggest that a combina- study, Honess and Jones (1937) suggested that etch mor- tion of electrostatic, stereochemical, and geometric rec- phology expresses the symmetry of both the substrate ognition processes occur between organic molecules and crystal and the solvent by comparing etch patterns ob- mineral surfaces during crystal growth (Mann 1988). Al- tained using optically inactive and active solvents on dif- though this hypothesis is largely drawn from ex situ SEM ferent crystallographic faces of carbonate minerals in de- examinations of crystals taken from growth solutions, it tail. Etching has also found applications in separating suggests an underlying principle: The geometry and enantiomorphous conglomerates and assigning the abso- chemistry of functional groups that characterize acidic lute con®guration of polar and chiral compounds (Shimon amino acids have highly specialized interactions with spe- et al. 1986). However, we are unaware of studies using ci®c surface site types. If true, it may be possible to un- dissolution or other chemical-etching methods to probe ravel the mechanisms governing ``recognition'' processes the mechanisms by which additives modify the relative in a systematic and predictable way. reactivities of surface sites with similar structures. In this dissolution study, we investigate the nature of Asp inter- Approach of this study actions with calcite surface sites by combining in situ With the development of ¯uid-cell techniques in atomic microscopic observations of variations in etch pit mor- force microscopy (AFM), we begin to determine the na- phologies developed in real time with ex situ examination ture of these specialized interactions through in situ ob- of surface topographies produced after long-term expo- 880 TENG AND DOVE: ASPARTATE INTERACTION WITH CALCITE sure to aspartate. We show that sites along speci®c crys- calibration curve. Scan rates ranged from 5 to 9 Hz with tallographic directions become less reactive during dis- 512 sampling points per scan line, which corresponds to solution in the presence of Asp and new surfaces that capture times of 1±2 min per image. The equilibrium im- were previously unstable are formed. These surfaces are aging temperature was approximately 30 ЊC because of the same as those developed in Asp-modi®ed growth ex- thermal energy created by the laser and microscope body periments. This discovery suggests that mineral dissolu- (Dove and Chermak 1994). Minimal thermal drift of the tion studies using suites of selected amino acids and or- scanner was observed. ganic compounds may help to unravel the mechanisms The ¯uid cell used in these AFM experiments had an by which organic compounds mediate the nucleation and internal volume of 50 ␮L and a ¯ow-through capability growth of minerals in biological systems. that permits solutions to pass continuously through the reaction chamber. See Dove and Chermak (1994) for a EXPERIMENTAL PROCEDURES description of cell design. The mounted samples were Sample and solution preparation ®rst imaged in air to locate a relatively ¯at area and to The calcite samples were obtained from a single large optimize image quality. Then solutions were manually in- crystal of optical-quality Iceland spar. Each experiment jected into the ¯uid cell using a syringe. After wetting began by cleaving a new fragment from this crystal to the cell and surfaces, a peristaltic pump was used to obtain a fresh (1014) surface. Fragments were handled maintain a constant ¯ow of solution through the cell at a with tweezers to avoid surface contamination by skin oils maximum pumping rate of 0.8 mL/h. and were cleaned with small bursts of N2(g) to remove small particles and dust adhering to the surfaces. Samples Treatment of the surface examined by ex-situ used in ¯uid cell AFM experiments had dimensions of dissolution approximately 5 ϫ 5 ϫ 1mm3. Each fragment was The samples prepared for long-term etching (see pre- mounted onto a steel puck using sealing wax. Larger sam- vious section) were placed in a ¯ow-through chamber ples with dimensions of 15 ϫ 15 ϫ 3mm3 were prepared with a ¯uid volume of 20 mL. Over a period of two for the macroscopic optical analyses. These fragments weeks, a peristaltic pump circulated 500 mL of 0.01 and were mounted on glass slides using sealing wax to cover 0.1 M Asp solutions through the chamber at a rate of 20 all faces but one for an exposed area of approximately mL/h. Using optical microscopy, progressive etching of 15 ϫ 15 mm2. the surface was monitored daily. At the end of this period, Aspartate (Asp) in the L-monosodium form [Na- the samples were removed from solution and dried with OOCCH CH(NH )COOH] was used in our experiments. 2 2 small bursts of N2 gas. Macroscopic observations of the The Asp molecule is a two-C chain that has a carboxyl resulting etched surfaces were examined using a Leica and an amine group (zwitterionic moiety) attached to one Quantimet Q-570 digital image analyzer coupled to an C and a carboxyl group attached to the other as a side optical microscope. Incident illumination was used to ob- chain. The carboxylate groups have pKa values of 1.99 tain optimal contrast of surface features and the images and 3.9 associated with the zwitterion and the side chain, were stored as digital ®les. respectively (Voet and Voet 1995). The pKa value of the amine group is approximately 10. Stock solutions of 10Ϫ2 OBSERVATIONS and 10Ϫ1 M Asp were prepared using bio-research grade Development of etch features in deionized water deionized water (resistivity greater than 18 megohm-cm, total organic C less than 10 ppb). These solutions were Within a few seconds of injecting deionized water into equilibrated with air before use and the pH of all solu- the ¯uid cell, AFM discerned the development of disso- tions was measured to be approximately 5.6. Thus, the lution etch pits with near-perfect rhombus symmetry on Asp molecule had fully ionized carboxylate groups and a the calcite (1014) face. Figure 1 shows a typical AFM protonated amine group at the pH of our experiments. image of a calcite surface exposed to distilled water for less than 2 min. Surfaces were relatively ¯at with a max- Imaging by ¯uid contact AFM imum topographic relief of approximately 30 AÊ . Individ- AFM experiments were conducted using a Nanoscope ual 3 AÊ steps were observed over the imaging area, and III scanning probe microscope (Digital Instruments) the terraces between adjacent steps were atomically ¯at. equipped with an E-sized piezoelectric scanner (maxi- The shape of these etch pits is consistent with previous mum scan area of 14 ϫ 14 ␮m2). Surfaces were imaged studies reporting the dissolution of calcite in water (Hill- using commercially available Si3N4 cantilevers having a ner at al. 1992a, 1992b; Stipp et al. 1994). triangular shape, length of 200 ␮m, and force constant of For each new experiment, we used the orientations of approximately 0.6 N/m. Radii of lever tips were approx- etch pits developed in water to establish the nanoscale imately 30±50 nm, which corresponds to a theoretical crystallographic orientation of fragments. Gratz et al. contact force of 27±46 ϫ 10Ϫ9 N between tip and sample (1993) and Stipp et al. (1994) have shown that pit edges (Eggleston 1994). To reduce the possibility of arti®cial (and thus step edges) correspond to the [441] and [481] changes in microtopography by tip-surface interactions, directions as illustrated in Figure 1. This orientation al- the interactive force was minimized by tuning the force lowed us to make direct comparisons with subsequent TENG AND DOVE: ASPARTATE INTERACTION WITH CALCITE 881

FIGURE 1. Fluid-cell AFM image of a (1014) calcite crystal FIGURE 2. Fluid-cell AFM image of the same area of the face reacting with deionized water shows the etch features pro- (1014) face shown in Figure 1 after exposure to 10Ϫ1 M Asp duced after two minutes of dissolution. The individual steps are solution for 10 min. The rhombus-shaped etch pits were removed monomolecular units having a height of 3 AÊ . The pit edges and during continued dissolution and triangular pits formed. The step edges reveal the [441] and [481] crystallographic directions three sides of the pits correspond to the [010], [451], and [411] that develop in simple inhibitor-free solutions and thus disclose directions and step heights are approximately 3 AÊ . the nanoscale orientation of the calcite fragment. The maximum topographic relief of this surface is about 30 AÊ . ures 3b and 3c show the evolution of surface structures observations of pit development with the introduction of that occurs with the introduction of aspartate. Figure 3b aspartate. was collected over an imaging time of 56 s (scan rate of 9 Hz) and records a sharp contrast between the water- Transition in etch pit morphology with introduction of dissolved surface (upper part of image) and Asp-reacted aspartate surface (lower part). The scratchy center portion of this After establishing the crystallographic orientation of image corresponds to a 14 s transition period during each new fragment in deionized water, the input solution which the solution was changed from 0 to 10Ϫ1 M Asp was changed to 10Ϫ2 or 10Ϫ1 M Asp. Within a few tens as a step input. This occurred while the cantilever was of seconds, the introduction of aspartate solutions pro- disturbed by the uneven ¯ow of injecting a pulse of the moted a sharp transition in the dissolution features to new solution. The last 18 s of Figure 3b (lower part of yield etch pits having a triangular morphology. Figure 2 image) recorded the early stage of triangular pit devel- illustrates the triangular surface structures that typically opment. Newly developed pits with sizes as small as a developed over 10 min of exposure to a 10Ϫ1 M aspartate few tens of nanometers already bore a triangular-like solution (for the same area shown in Fig. 1). None of the shape, but the directions of step edges were irregular. Fig- three step edges forming these triangular pits was parallel ure 3c shows that, after another minute, the [441] and to the pit edge directions observed in Figure 1. Rather, [481] crystallographic directions that were stable during they revealed new crystallographic directions: [010], dissolution in water were hardly discernible. Yet, only [451], and [411]. The edge lengths of triangles shown in two of the three triangular pit edges, [451] and [411], had Figure 2 varied from a few to 500 nm, illustrating that clearly developed during this period. A comparison of the pit morphology was not a function of pit size. The Figures 3b and 3c showed that while Asp promotes a angles between step edges composing the pits are ap- transition in dissolution surface structure within seconds, proximately 64Њ between [451] and [411], and 58Њ be- the rate of development of the [010] direction is slower. tween [451] and [010], and [411] and [010] directions. Rather, this third edge continued to display the two com- Figures 3a±3d record the transition from rhomboid to ponents of the previously dominant [441] and [481] di- triangular pits that resulted when the input solution was rections, and the [010] edge was still not fully stabilized changed from a 0 to 10Ϫ1 M Asp solution. Figure 3a after another two minutes as indicated in Figure 3d, shows the characteristic etch features produced during which shows the almost fully developed triangular pits. short-term exposure to distilled water. Intermediate Fig- In repeated experiments, we found that the transition from 882 TENG AND DOVE: ASPARTATE INTERACTION WITH CALCITE

FIGURE 3. A time sequence of AFM images showing the input solution was changed. (c) Time ϭ 5 min. The [451] and transition from rhombus to triangular etch-pit morphology. Each [411] directions have clearly developed, but triangular form is image was collected over a 56 s period. (a) Time ϭ 1 min. The incomplete. (d) Time ϭ 7 min. Triangular morphology is nearly (1014) face in deionized water results in formation of rhom- complete but the pit edges corresponding to the [010] direction bus-shaped etch pits. (b) Time ϭ 4 min. Transition from deion- continue to have curvature. Incomplete development of the [010] ized water (upper portion of image) to 10Ϫ1 M Asp solution (low- relative to the [451] and [411] directions suggests that surface er portion of image) shows that Asp immediately interacts with sites along [010] have a weaker interaction with Asp or perhaps the calcite surface to modify etch-pit morphologies. The central form at a slower rate. Continued reaction to formation of ``scratchy'' portion of image corresponds to the 14 s period when the isosceles features shown in Figure 2.

a rhombus to triangular etch-pit morphology was readily angular pits. These pits were so extensive that intercon- reversible when the input solution was returned to water. nected tetrahedral etch hillocks had developed as residual features from the formerly ¯at (1014) cleavage surface. Development of etch hillocks with long term exposure to Figure 4 shows a typical surface that developed over an aspartate solutions area of approximately 3 ϫ 3mm2. Sizes of the hillocks, Examining the calcite samples that were continuously as measured by the side length of their base triangles, exposed to 0.1 M Asp solutions in a ¯ow-through cham- ranged from a few micrometers to several hundred ber for two weeks also revealed the development of tri- micrometers. TENG AND DOVE: ASPARTATE INTERACTION WITH CALCITE 883

tion was ®rst reported in the macroscopic etch experi- ments by Honess and Jones (1937). However, we are only beginning to understand Asp-calcite surface interactions. The mechanism by which aspartate promotes the de- velopment of {0001} and {1101} forms can be under- stood in terms of the calcite structure and the nature of aspartate-surface interactions. First, the of calcite (point group 32/m) is often described as alter-

nating planes of Ca atoms and CO3 groups. These planes are perpendicular to the c axis (e.g., Fig. 5) and constitute the basal {0001} form. Individual carbonate groups with- in a plane are also perpendicular to the c axis, but they are rotated in adjacent carbonate planes in a staggered conformation. Thus, only the basal {0001} faces bear a threefold rotational symmetry. For a detailed description of the calcite structure see Reeder (1983) and references therein. The {0001} is also unusual in comparison to oth- er forms because it possesses a strong electrical dipolar moment induced by single component layers (pure Ca or

pure CO3). Because the charge on (0001) faces cannot be fully neutralized by underlying atoms (of opposite charge), surfaces consisting of the {0001} form are less stable than those faces that lack this dipolar character. This lack of stability is in agreement with the theoretical analysis of Titiloye et al. (1993). Second, the ability of aspartate to modify calcite sur- face site reactivities can be examined in terms of the rel- ative surface complexation strength of ligands in solution.

In the presence of H2O, Stipp and Hochella (1991) and Van Cappellen et al. (1993) have shown that surface- bound Ca atoms form ϾCa-OH complexes by a reaction that can be represented as: FIGURE 4. Digital image of etch hillock features developed ϩ ↔ ϩ on the (1014) face after etching in 10Ϫ1 M Asp solution for two ϾCa ϩ H2O ϾCa-OH ϩ H (1) weeks. The new surface is characterized by well-oriented tetra- where ``Ͼ`` indicates a surface-bound Ca atom. With the hedral etch hillocks that are interconnected by triangular etch introduction of aspartate, Reaction 1 competes with the pits. The labels show the directions of the three component faces ϾCa-Asp complexation reaction: composing the tetrahedrons and the directions of individual lay- ers composing the hillocks. The corresponding crystallographic ϾCaϩ ϩ AspϪ ↔ ϾCa-Asp. (2) axes are also shown. Although the complexation constant for surface Reaction 2 is unknown, we infer that the binding constant of Re- Because the fragments used in these ex situ dissolution action 2 is signi®cantly greater than that of Reaction 1 experiments were suf®ciently large to determine the crys- by analogy with the relative strengths of comparable tallographic orientation mechanically, we were able to as- aqueous complexation constants (e.g., Schindler and certain the crystallographic directions of the hillock Stumm 1987). The aqueous complexation reaction: slopes. Using image analysis, we found that the slopes of Ca2ϩ ϩ Asp ↔ Ca2ϩ-Asp(aq) (3) each hillock corresponded to the calcite (0001), (1101), and Ê (0111) faces. Thus, the 3 A steps, which represent the mon- has a relatively strong pKa of 1.6 (Chaberek and Martell omolecular steps of the original (1014) face, were the tri- 1959) compared to the association of H2O with Ca ion angular layers within each hillock. These layers intersect the by the reaction: (0001) face at the [010] axis, the (1101) face at the [451] Ca2ϩ ϩ H O ↔ Ca-OH(aq) ϩ Hϩ (4) axis, and the (0111) face at the [411] axis. Recall that these 2 directions are parallel to pit edges observed in the AFM which has a pKa value of 12.85 (e.g., Stumm and Morgan experiments. 1981). Thus, the pKa value of Reaction 3 is approximately ten orders of magnitude greater than that of Reaction 4, DISCUSSION which suggests that the ϾCa-Asp complex in Reaction 2 The formation of triangular etch pits on the cleavage may also be the predominant surface species when as- rhombohedron of calcite by contact with aspartate solu- partate molecules are present in solution. This is signi®- 884 TENG AND DOVE: ASPARTATE INTERACTION WITH CALCITE

solely by electrostatic interactions of the lone carboxyl group with the pure Ca layer. The other carboxyl of the zwitterion is less likely to play a role because its negative ϩ charge is partially neutralized by the adjacent NH3 group. Our ®ndings and the study of Rajam and Mann (1990) indicate that stereochemical and geometric recognition mechanisms do not play a signi®cant role in {0001} development. Mechanism for development of the {1101} form The orientations of residual hillocks in Figure 4 show FIGURE 5. Schematic cross-sectional view of the calcite structure showing the crystallographic forms developed in the that Asp also stabilizes the equivalent (1101) and (0111) presence of Asp from the initial {1014} cleavage surface. The faces. In contrast to the basal {0001} form, these faces {0001} and {1102} forms are single component layers of pure are composed of equal numbers of Ca and CO3 groups

Ca atoms or CO3 groups but differ from each other in the ori- and hence are not dipolar. The mechanism for the devel- entation of planar CO3 groups and atomic spacings (see discus- opment of this form is not readily apparent as adjacent sion). In contrast, the {1100} form has equal numbers of Ca and cation and anion sites within the (1101) and (0111) planes CO3. Solid lines indicate the {0001} and {1101} forms that be- are separated by nearly 8 AÊ to result in a low surface- come stabilized upon the introduction of Asp to develop the tet- packing density. Also, adjacent Ca sites in the planes are rahedral etch hillocks shown in Figure 4. Dashed lines represent separated by nearly 16 AÊ . Each of these distances is great- the combination of {1102} and {1100} forms to yield the pseudo er than the length of single aspartate molecule, which {1101} form. suggests that neither face is stabilized by the binding of carboxyl groups to surface sites in these planes. Thus, cant because the stronger interaction of Asp with the sur- crystallographic and organic stereochemical constraints face can inhibit the hydrolysis of ϾCa sites during indicate that pure (1101) and (0111) faces cannot form. dissolution (see later discussion) by at least two mecha- These considerations lead us to propose that the (1101) nisms: (1) The larger physical size of the aspartate mol- and (0111) faces that occur as two slopes forming the ecules in comparison with inorganic aqueous ions can macroscopic residual hillocks in Figure 4 are the result limit the approach of H2O molecules to the hydrolyzing of stabilizing alternating segments of the (1102) and calcite-water interface (e.g., Conway 1981). (2) The for- (1100), and the (0112) and (0110) faces at a microscopic mation of a ϾCa-Asp surface complex reduces the rate scale. The (1102) and (0112) faces belong to the {1102} or frequency of hydrolysis reactions (e.g., see discussion form of calcite. Similar to {0001}, faces in {1102} are in Dove and Czank 1995, and references therein). Hence, also composed of pure Ca (or pure CO3) layers. Stabili- portions of the calcite surface that are Ca rich or struc- zation of this form by Asp can be attributed to the same turally well-matched to the aspartate stereochemistry are electrostatic mechanism that promotes development of protected or preferentially stabilized by Asp. the basal face (see previous section). However, extensive development of {1102} does not appear to be permitted. Mechanism for development of the {0001} form This may be understood by comparing the structure of As discussed earlier, the {0001} form does not develop the {0001} and {1102} forms. The latter has a higher in simple aqueous solutions but rather becomes stabilized density of Ca atoms or CO3 groups composing the surface with the introduction of Asp. Expression of {0001} on layer (and thus, a still-higher dipole moment). For ex- the (1014) cleavage face is necessarily constrained by the ample, the schematic illustration in Figure 5 shows that orientation of its pure Ca layer with respect to the dis- the distance between adjacent Ca atoms in the {1102} solution surface. Figures 2 and 3 show that within each form is only 3.83 AÊ compared to approximately 4.99 AÊ 3AÊ layer on the (1014) face, one side of each triangular for the {0001} form. This results in a Ca surface density pit forms by preferentially stabilizing sites lying along the that is 70% greater. The result of this greater dipole mo- [010] direction. The [010] direction is signi®cant because ment is to limit stabilization of {1102} to small areal it is the line formed by the intersection of the cleavage scales. As seen in Figure 5, crystallographic structure dic- surface plane with the single component layer of Ca at- tates that the shortest segment of atoms that can form Ê oms or CO3 groups composing the {0001} form. As Asp along this plane is approximately 8 A long. binds with Ca atoms exposed along the [010] direction, Although our investigation cannot estimate a critical one side of each pit becomes a ``stack of edges'' parallel length (or area) above which {1102} is destabilized, one to [010]. Continued dissolution leads to a (0001) face as can see that the dipole moment associated with this form the pit deepens. With extensive dissolution, the pits en- can be reduced by the formation of the intermittent pris- large and merge to form the residual hillocks illustrated matic face segments illustrated in Figure 5. Unlike in Figure 4. One slope on each hillock corresponds to the {0001} and {1102}, the faces composing the prismatic {0001} form. {1100} form are not dipolar. However, previous studies This mechanism suggests that Asp stabilizes {0001} propose that a combination of electrostatic, geometric, TENG AND DOVE: ASPARTATE INTERACTION WITH CALCITE 885 and stereochemical recognition processes between calcite tively charged Asp molecules was insuf®cient to modify surfaces and Asp molecules can stabilize the prismatic the hydrolysis of surface sites on {0001} relative to sites form. Mann et al. (1990) stated that recognition occurs on the {1014} faces. by cooperative electrostatic interactions of the carboxyl One possible means of fully stabilizing the {1102} fac- and amine groups of the zwitterion with ϾCa atoms and es would be the use of a still-higher concentration of Asp.

ϾCO3 groups in adjacent layers of the {1100} form. However, we were not able to test this without exceeding These surface groups make unique bidentate binding sites the solubility of the calcium aspartate salt. Another pos- because the cations and anions are separated by 3.3 AÊ . sibility is the use of a compound that has additional rec- This is equal to the distance between the amino and car- ognition properties between the organic and inorganic boxyl groups of the Asp zwitterionic moiety components at the interface. This was accomplished by ϩ Ϫ ( H3NCH2COO ). This matching also allows suf®cient Berman et al. (1995) by nucleating calcite onto a sub- space to position the ␣-C near the surface because CO3 strate matrix of 10,12-pentacosadiynoic acid (a 25-C groups on the {1100} form are oriented so that only one chain carboxylic acid). Their observation of {1102} for- O atom ``protrudes'' (Mann et al. 1990). This binding mation led to a model using cooperative alignment of Ϫ con®guration enables the remaining -CH2COO group to calcite components with carboxyl groups at the mineral- replace a CO3 position in the adjacent anion layer. The organic interface in addition to electrostatic interactions overall geometric and stereochemical cooperativity re- with the ϾCaϩ surface sites. sults in Asp binding on the {1100} calcite faces in a linear pattern. We suggest that development of {1100} Signi®cance to understanding interactions of other during dissolution in the presence of Asp occurs by the amino acids with calcite same recognition processes proposed by Mann et al. Our ®ndings suggest that amino acids with chemical (1990). and physical structures similar to aspartate may modify Our AFM images have insuf®cient microscopic reso- calcite reactivities in growth and dissolution by similar lution to prove that (1101) and (0111) are a combination mechanisms. Glutamate is the only naturally occurring of the corresponding faces in the {1102} and {1100} amino acid that shares the acidic character of Asp. A forms by direct observation. For example, Figure 2 shows macroscopic study by Honess and Jones (1937) demon- a 5.86 ϫ 5.86 ␮m2 area as a composite of 512 ϫ 512 strated that Glu also affects the etch morphology of pixels of data. Thus, each pixel covers an area of nearly calcite. 13 023 AÊ 2 or a length of approximately 114 AÊ , which is This paper is not intended to address the kinetics of 10±15 times greater than the length of segment repre- calcite dissolution in Asp solutions, but we can make sented in Figure 5. However, two lines of experimental qualitative observations regarding the in¯uence of Asp on evidence support this model. First, only the projection dissolution and growth rates. First, we see that the inter- of the {0001} and {1101} forms onto the (1014) face actions of Asp with calcite do not render surface sites can explain the isosceles triangular features shown in nonreactive. Sites along the [010], [451], and [411] di- Figure 2. This projection yields angles of 63Њ,58Њ, and rections become less reactive relative to those along other 58Њ between the three step edges, which is consistent with directions. Yet, Figure 3 shows that step edges visibly our experimental observations. The previous discussion retreat in Asp-bearing solutions at a rate of several hun- indicates that the likely explanation for development of dred nanometers per minute. We predict that rates of {1101} is an average expression of the {1100} and growth or dissolution in comparable Glu and Asp solu- {1102} forms. A second piece of evidence is suggested tions should be similar because of their similar interaction by examining the etch hillock slopes in Figure 4. In con- strengths. Second, the continued migration of steps sug- trast with the very smooth (0001) faces expressed on the gests that Asp is a relatively weak inhibitor compared to hillocks, the (1101) and (0111) faces are uneven as evi- phosphate ion, a well-documented strong inhibitor. AFM denced by the alternating light-dark patterns. This sug- observations of Dove and Hochella (1993) showed that gests that neither of these surfaces are true crystallo- all step migration ceases with the introduction of a 10 3Ϫ 3 graphic faces. ␮M PO4 solution. Thus, bulk dissolution rates in PO4 - Our ®ndings support the previous study by Titiloye et bearing solutions should be signi®cantly slower than in al. (1993) indicating that the magnitude of the dipole mo- Asp solutions. ment on a crystal face is central to modifying electrostatic Although amino acids are simplistic analogs for the interactions and stabilizing individual faces. We postulate complex proteins present in organic-rich environments, that a still-stronger driving force is needed to overcome our observations suggest that a systematic growth or dis- the greater dipole moment on the {1102} form to slow solution study using a suite of selected amino acids could the hydrolysis rate of groups within this plane and fully be used to unravel the mechanisms by which their func- stabilize the formation of the corresponding (1102) and tional groups interact with surfaces and allow us to quan- (0112) faces expressed by the hillocks. This is supported tify their controls on growth or dissolution kinetics. By by our experimental observations that {0001} did not de- investigating the in¯uence of neutral side chains, polar velop when Asp concentrations were Յ10Ϫ2 M. At lower uncharged side chains, charged side chains, and others in concentrations, we speculate that the quantity of nega- simple systems, we may develop a mechanistic frame- 886 TENG AND DOVE: ASPARTATE INTERACTION WITH CALCITE work for solute-surface interactions that govern the for- Addadi, L., Moradian, J., Shay, E., Maroudas, N.F., and Weiner, S. (1987) mation of different mineral polymorphs and their mor- A chemical model for the cooperation of sulfates and carboxylates in calcite crystal nucleation: relevance to biomineralization. Proceedings phologies in biological systems. of the National Academy of Sciences 84, 2732±2736. Berman, A., Addadi, L., and Weiner, S. (1988) Interactions of sea-urchin Determining surface controls of other inhibitors on etch skeleton macromolecules with growing calcite crystals: A study of in- morphology tracrystalline proteins. Nature, 331, 546±549. It is generally accepted that the shapes and orientations Berman, A., Hanson, J., Leiserowitz, L., Koetzle, T.F., Weiner, S., and of etch pits developed on crystallographic faces corre- Addadi, L. (1993) Biological control of crystal texture: a widespread strategy for adapting crystal properties to function. Science, 259, 776± spond to the projection of the point group symmetry of 779. that crystal onto the individual crystal face concerned Berman, A., Ahn, D.L., Lio, A., Salmeron, M., Reichert, A., and Charych, (e.g., Zoltai and Stout 1984; Sangwal 1987; Putnis 1992). D. (1995) Total alignment of calcite at acidic polydiacetylene ®lms: Our ®ndings show that this rule-of-thumb holds even in cooperativity at the organic-inorganic interface. Science, 269, 515±518. solutions in which specialized surface interactions modify Chaberek, S., and Martell, A.E. (1959) Organic sequestering agents. Wi- the etch morphology. More speci®cally, the glide plane ley, New York. Crenshaw, M.A. (1972) The soluble matrix from Mercenaria mercenaria symmetry on the (1014) face is unaffected by the change shell. In H. K. Erben, Ed., Biomineralization research reports: Inter- in activity of surface sites. However, our results demon- national Symposium on Problems in Biomineralization 6, p. 6±11. strate that the sites that control the development of the Schattaure Verlag, Stuttgart. [411] and [481] directions in water become less reactive Conway, B.E. (1981) Ionic Hydration in Chemistry and Biophysics. Stud- compared to those sites that lead to the formation of ies in Physical and Theoretical Chemistry 12, 773 p. Elsevier, Amsterdam. [451], [411], and [010] directions when Asp is intro- Dove, P.M. and Chermak, J. (1994) Mineral-water interactions: Fluid cell duced. This suggests that the stability of steps along par- applications of scanning force microscopy. In K.L. Nagy and A.E. ticular crystallographic directions is controlled by the rel- Blum, Eds., CMS Workshop Lectures: Scanning Probe Microscopy of ative reactivities of surface sites. Thus, we show that Clay Minerals, 139±170 p. Clay Minerals Society, Washington, DC. compounds having specialized interactions with the sur- Dove, P.M. and Czank, C.A. (1995) Crystal chemical controls on the dis- solution kinetics of the isostructural sulfates: Celestite, anglesite, and face can yield new insights into controls on mineral barite. Geochimica et Cosmochimica Acta, 59, 1907±1915. reactivity. Dove, P.M. and Hochella, M.F., Jr. (1993) Calcite precipitation mecha- nisms and inhibition by orthophosphate: In situ observations by Scan- Dissolution experiments as a tool in biomineralization ning Force Microscopy. Geochimica et Cosmochimica Acta, 57, 705± studies 714. The results of this dissolution study are consistent with Eggleston, C.M. (1994) High-resolution scanning probe microscopy: Tip- surface interaction, artifacts, and applications in mineralogy and geo- previous growth experiments showing that Asp causes chemistry. In K.L. Nagy and A.E. Blum, Eds., Scanning Probe Mi- preferential development of the {0001} and possibly the croscopy of Clay Minerals. CMS workshop lectures, p. 3±90. Clay Min- {1100} forms of calcite (e.g., Addadi and Weiner 1985; erals Society, Washington, DC. Mann et al. 1990). This suggests that dissolution experi- Gratz, A.J., Hillner, P.E., and Hansma, P.K. (1993) Step dynamics and ments may offer an alternative means of identifying the spiral growth on calcite. Geochimica et Cosmochimica Acta, 57, 491± crystal face that forms and a more direct means of iden- 495. Hillner, P.E., Manne, S., Gratz, A.J., and Hansma, P.K. (1992a) AFM im- tifying those sites involved in stabilizing each new face. ages of dissolution and growth on a calcite crystal. Ultramicroscopy, Dissolution experiments have the advantages of being 42±44, 1387±1393. less dif®cult and less time-consuming to conduct than Hillner, P.E., Gratz, A.J., Manne, S., and Hansma, P.K. (1992b) Atomic growth studies. Yet, further study is needed to determine scale imaging of calcite growth and dissolution in real time. Geology , if etching features produced in the presence of inhibitors 20, 359±362. Honess, A.P. and Jones, J.R. (1937) Etch ®gure investigations with opti- during dissolution can be general indicators of the faces cally active solvents. Bulletin of the Geological Society of America, that become stabilized during mineral growth. If future 667±722. studies ®nd this holds true, dissolution studies may be- Lowenstam, H.A. (1981) Minerals formed by organisms. Science, 211, come a useful tool to determine the site-speci®c interac- 1126. tions of many different biological compounds with min- Lowenstam, H.A. and Weiner, S. (1989) On Biomineralization, p. 21±24. eral surfaces and to improve our understanding of the Oxford University Press, Oxford. Mann, S. (1988) Molecular recognition in biomineralization. Nature, 332, physical processes governing biomineralization. 119±124. (1993). Molecular tectonics in biomineralization and biomimetic ACKNOWLEDGMENTS materials chemistry. Nature, 365, 499±505. This work was supported by the Basic Energy Sciences Program of the Mann, S., Didymus, J.M., Sanderson, N.P., Heywood, B.R., and Aso Sam- U.S. Department of Energy through grant number DE-FG05-95-ER14517. per, E.J. (1990) Morphological in¯uence of functionalized and non- We thank Dirk Bosbach, Kathryn Nagy, and an anonymous reviewer for functionalized ␣, ␻-dicarboxylates on calcite crystallization. Journal helpful comments during review. We also thank Rich Reeder for careful Chemical Society Faraday Transaction, 86, 1873±1880. review of calcite crystallography during the ®nal editorial handling. 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