Implications for Biomineralization
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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 mineral 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 minerals 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 lead 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) cleavage 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 b-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 a-aminosuccinate (aspartate) and g-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