Purification,characterization, and in vitro mineralization studies of a novel goose eggshell matrix protein,ansocalcin

Biomineralization is an important process in which hard tissues are generated through mineral deposition, often assisted by biomacromolecules. Eggshells, because of their rapid formation via mineralization, are chosen as a model for understanding the fundamentals of biomineralization. This report discusses purification and characterization of various proteins and peptides from goose eggshell matrix. A novel 15-kDa protein (ansocalcin) was extracted from the eggshell matrix, purified, and identified and its role in mineralization evaluated using in vitro crystal growth experiments. The complete amino acid sequence of ansocalcin showed high homology to ovocleidin-17, a chicken eggshell protein, and to C-type lectins from snake venom. The amino acid sequence of ansocalcin was characterized by the presence of acidic and basic amino acid multiplets. In vitro crystallization experiments showed that ansocalcin induced pits on the rhombohedral faces at lower concentrations (<50 microg/ml). At higher concentrations, the nucleation of calcite crystal aggregates was observed. Molecular weight determinations by size exclusion chromatography and sodium dodecyl sulfate -polyacrylamide gel electrophoresis showed reversible concentration-dependent aggregation of ansocalcin in solution. We propose that such aggregated structures may act as a template for the nucleation of calcite crystal aggregates. Similar aggregation of calcite crystals was also observed when crystallizations were performed in the presence of whole goose eggshell extract. These results show that ansocalcin plays a significant role in goose eggshell calcification.     

Eggshell matrix protein mimics: designer peptides to induce the nucleation of calcite crystal aggregates in solution

Ansocalcin is a novel goose eggshell matrix protein with 132 amino acid residues, which induces the formation of polycrystalline calcite aggregates in in vitro crystallization experiments. The central region of ansocalcin is characterized by the presence of multiplets of charged amino acids. To investigate the specific role of charged amino acid multiplets in the crystal nucleation, three short peptides REWD-16, REWDP-17 (containing charged doublets), and RADA-16 (alternating charged residues) were synthesized and characterized. The aggregation of these peptides in solution was investigated using circular dichroism, intrinsic tryptophan fluorescence, and dynamic light scattering experiments. The peptides REWD-16 and REWDP-17 induced the polycrystalline calcite crystal aggregates, whereas RADA-16 did not induce significant changes in calcite crystal morphology or aggregate formation in in vitro crystallization experiments. The lattice and morphology of the calcite crystals were characterized using X-ray diffraction and scanning electron microscope. The results discussed in this paper reveal the importance of multiplets of charged amino acid residues toward the nucleation of polycrystalline calcite crystal aggregates in solution.     

Structure-function relationship of avian eggshell matrix proteins: a comparative study of two major eggshell matrix proteins, ansocalcin and OC-17

The role of individual matrix proteins in avian eggshell calcification is poorly understood despite numerous attempts to characterize and localize their presence in the eggshell matrix. Ansocalcin, the major matrix protein from goose eggshell, was found to induce the formation of calcite crystal aggregates under in vitro. Owing to its high similarity with the chicken eggshell matrix protein ovocleidin 17 (OC-17), a comparative investigation has been carried out to understand the structure-function relationship. RP-HPLC shows that ansocalcin is the major component in extracts of goose eggshells before and after bleach treatment. However, OC-17 was observed in minute quantities in the extract of bleach-treated chicken eggshells. In vitro crystal growth experiments showed that OC-17 and ansocalcin interact differently with the calcite crystals formed. Circular dichroism, intrinsic tryptophan fluorescence, and dynamic light scattering studies showed that, under the conditions used in our experiments, OC-17 does not aggregate in solution or induce the nucleation of calcite aggregates in the concentration range used. These observations indicate that OC-17 and ansocalcin play different roles in the eggshell calcification. To our knowledge, this is the first report on the comparison of properties of homologous eggshell proteins that belong to the same phylogeny.     

Secretion pattern, ultrastructural localization and function of extracellular matrix molecules involved in eggshell formation

The chicken eggshell is a composite bioceramic containing organic and inorganic phases. The organic phase contains, among other constituents, type X collagen and proteoglycans (mammillan, a keratan sulfate proteoglycan, and ovoglycan, a dermatan sulfate proteoglycan), whose localization depends on a topographically defined and temporally regulated deposition. Although the distribution of these macromolecules in the eggshell has been well established, little is known about their precise localization within eggshell substructures and oviduct cells or their pattern of production and function during eggshell formation. By using immunofluorescent and immuno-ultrastructural analyses, we examined the distribution of these macromolecules in oviduct cells at different post-oviposition times. To understand the role of proteoglycan sulfation on eggshell formation, we studied the effects of inhibition of proteoglycan sulfation by treatment with sodium chlorate. We showed that these macromolecules are produced by particular oviduct cell populations and at precise post-oviposition times. Based on the precise ultrastructural localization of these macromolecules in eggshell substructures, the timing of the secretion of these macromolecules by oviduct cells and the effects on eggshell formation caused by the inhibition of proteoglycan sulfation, the putative role of mammillan is in the nucleation of the first calcite crystals, while that of ovoglycan is to regulate the growth and orientation of the later forming crystals of the chicken eggshell.     

Colloidal-gold immunocytochemical localization of osteopontin in avian eggshell gland and eggshell.

During mineralization of the avian eggshell, there is a sequential and orderly deposition of both matrix and mineral phases. Therefore, the eggshell is an excellent model for studying matrix-mineral relationships and the regulation of mineralization. Osteopontin, as an inhibitor of crystal growth, potently influences the formation of calcium phosphate and calcium carbonate biominerals. The purpose of this study was to characterize matrix-mineral relationships, specifically for osteopontin, in the avian eggshell using high-resolution transmission (TEM) and scanning (SEM) electron microscopy to gain insight into how calcite crystal growth is structured and compartmentalized during eggshell mineralization. Osteopontin was localized at the ultrastructural level by colloidal-gold immunocytochemistry. In EDTA-decalcified eggshell, an extensive matrix network was observed by TEM and SEM throughout all regions and included interconnected fibrous sheets, irregularly shaped aggregates, vesicular structures, protein films, and isolated protein fibers. Osteopontin was associated with protein sheets in the highly mineralized palisades region; some of these features defined boundaries that compartmentalized different eggshell structural units. In fractured and undecalcified eggshell, osteopontin was immunolocalized on the {104} crystallographic faces of calcite-its natural cleavage plane. The specific occlusion of osteopontin into calcite during mineralization may influence eggshell structure to modify its fracture resistance.     

Ultrastructural matrix-mineral relationships in avian eggshell, and effects of osteopontin on calcite growth in vitro

We investigated matrix–mineral relationships in the avian eggshell at the ultrastructural level using scanning and transmission electron microscopy combined with surface-etching techniques to selectively increase topography at the matrix–mineral interface. Moreover, we investigated the distribution of osteopontin (OPN) in the eggshell by colloidal-gold immunolabeling for OPN, and assessed the effects of this protein on calcite crystal growth in vitro. An extensive organic matrix network was observed within the calcitic structure of the eggshell that showed variable, region-specific organization including lamellar sheets of matrix, interconnected fine filamentous threads, thin film-like surface coatings of proteins, granules, vesicles, and isolated proteins residing preferentially on internal {1 0 4} crystallographic faces of fractured eggshell calcite. With the exception of the vesicles and granules, these matrix structures all were immunolabeled for OPN, as were occluded proteins on the {1 0 4} calcite faces. OPN inhibited calcite growth in vitro at the {1 0 4} crystallographic faces producing altered crystal morphology and circular growth step topography at the crystal surface resembling spherical voids in mineral continuity prominent in the palisades region of the eggshell. In conclusion, calcite-occluded and interfacial proteins such as OPN likely regulate eggshell growth by inhibiting calcite growth at specific crystallographic faces and compartmental boundaries to create a biomineralized architecture whose structure provides for the properties and functions of the eggshell.     

A novel extrapallial fluid protein controls the morphology of nacre lamellae in the pearl oyster, Pinctada fucata

Mollusk shell nacre is known for its superior mechanical properties and precisely controlled biomineralization process. However, the question of how mollusks control the morphology of nacre lamellae remains unresolved. Here, a novel 38-kDa extrapallial fluid (EPF) protein, named amorphous calcium carbonate-binding protein (ACCBP), may partially answer this question. Although sequence analysis indicated ACCBP is a member of the acetylcholine-binding protein family, it is actively involved in the shell mineralization process. In vitro, ACCBP can inhibit the growth of calcite and induce the formation of amorphous calcium carbonate. When ACCBP functions were restrained in vivo, the nacre lamellae grew in a screw-dislocation pattern, and low crystallinity CaCO(3) precipitated from the EPF. Crystal binding experiments further revealed that ACCBP could recognize different CaCO(3) crystal phases and crystal faces. With this capacity, ACCBP could modify the morphology of nacre lamellae by inhibiting the growth of undesired aragonite crystal faces and meanwhile maintain the stability of CaCO(3)-supersaturated body fluid by ceasing the nucleation and growth of calcite. Furthermore, the crystal growth inhibition capacity of ACCBP was proved to be directly related to its acetylcholine-binding site. Our results suggest that a "safeguard mechanism" of undesired crystal growth is necessary for shell microstructure formation.     

Immunohistochemical localization of carbonic anhydrase isoenzymes I and II in human bone,cartilage and giant cell tumor

Summary Carbonic anhydrase isoenzymes I and II have been localized in human bone and cartilage. Osteoclasts are strongly positive for carbonic anhydrase II but very little if any reaction is observed for carbonic anhydrase I. In tendon giant cell tumor osteoclastlike-giant cells contained high amounts of carbonic anhydrase II suggesting the close relation of these cells to normal osteoclasts. In growth plate cartilage strong staining was obtained in late proliferative and hypertrophic chondroxytes as well as in extracellular matrix of hypertrophic zone also only with anti-human carbonic anhydrase II.     

Amelogenins: assembly, processing and control of crystal morphology.

The remarkable properties of enamel crystals and their arrangements in an extraordinary micro-architecture are clear indications that the processes of crystal nucleation and growth in the extracellular matrix are highly controlled. The major extracellular events involved in enamel formation are: (a) delineation of space by the secretory ameloblasts and the dentino-enamel junction; (b) self-assembly of amelogenin proteins to form the supramolecular structural framework; (c) transportation of calcium and phosphate ions by the ameloblasts resulting in a supersaturated solution; (d) nucleation of apatite crystallites; and (e) elongated growth of the crystallites. Finally, during the 'maturation' step, rapid growth and thickening of the crystallites take place, which is concomitant with progressive degradation and eventual removal of the enamel extracellular matrix components (mainly amelogenins). This latter stage during which physical hardening of enamel occurs is perhaps unique to dental enamel. We have focused our in vitro studies on three major extracellular events: matrix assembly, matrix processing and control of crystal growth. This paper summarizes current knowledge on the assembly, processing and effect on crystal morphology by amelogenin proteins. The correlation between these three events and putative functional roles for amelogenin protein are discussed.     

Selective precipitation and purification of monovalent proteins using oligovalent ligands and ammonium sulfate

This paper describes a method for the selective precipitation and purification of a monovalent protein (carbonic anhydrase is used as a demonstration) from cellular lysate using ammonium sulfate and oligovalent ligands. The oligovalent ligands induce the formation of protein-ligand aggregates, and at an appropriate concentration of dissolved ammonium sulfate, these complexes precipitate. The purification involves three steps: (i) the removal of high-molecular-weight impurities through the addition of ammonium sulfate to the crude cell lysate; (ii) the introduction of an oligovalent ligand and the selective precipitation of the target protein-ligand aggregates from solution; and (iii) the removal of the oligovalent ligand from the precipitate by dialysis to release the target protein. The increase of mass and volume of the proteins upon aggregate formation reduces their solubility, and results in the selective precipitation of these aggregates. We recovered human carbonic anhydrase, from crude cellular lysate, in 82% yield and 95% purity with a trivalent benzene sulfonamide ligand. This method provides a chromatography-free strategy of purifying monovalent proteins--for which appropriate oligovalent ligands can be synthesized--and combines the selectivity of affinity-based purification with the convenience of salt-induced precipitation.     

Partial biochemical and immunochemical characterization of avian eggshell extracellular matrices.

There is evidence to suggest that extracellular matrix molecules, such as proteoglycans, are involved in the regulation of mineral deposition in calcifying tissues. One mineralizing system which is characterized by extremely rapid mineralization is the hen eggshell. This eggshell consists of a pair of nonmineralized eggshell membranes subjacent to the calcified eggshell proper; the eggshell proper is organized into palisades (columns) of mineralized matrix separated by pores. Between the membranes and the shell proper are compacted foci of tissue called mammillary knobs, which are thought to be sites where mineralization is initiated. Previous work from this laboratory has shown the presence of types I, V, and X collagen in the shell membranes. To address the question of the possible role of proteoglycans and glycosaminoglycans in mineralization of the eggshell, two approaches were used. First, immunohistochemistry was performed with monoclonal antibodies to various proteoglycan and glycosaminoglycan epitopes. This analysis indicates that different glycosaminoglycans are localized to discrete regions within the eggshell. Dermatan sulfate is present within the matrix of the shell proper and, to a lesser extent, the mammillary knobs and the outer portion of the shell membranes. In contrast, keratan sulfate is found in the shell membranes and prominently in the mammillary knobs. Interestingly, different keratan sulfate antibodies immunostain distinct regions of the eggshell, which suggests that various types of keratan sulfate are distributed differently. The second approach utilized was to extract the eggshell membranes and recover anionic molecules by anion-exchange chromatography. This resulted in the extraction of material which was recognized by antibodies to keratan sulfate, but not to chondroitin sulfate. This material was very large, as evidenced by its elution in the void volume of a Sepharose CL-2B column. The large size may be due to the extensive cross-links known to occur in the eggshell. If eggshell membranes are extracted at elevated temperature, the material recovered is of much smaller size. These results indicate that molecules recognized by antibodies to glycosaminoglycans are present in the eggshell, and their localized distribution relative to the calcified matrix suggests that they may be involved in the regulation of mineral deposition.     

Aspects of shell formation in eggs of the tuatara,Sphenodon punctatus

The flexible shell from eggs of the tuatara (Sphenodon punctatus) is comprised of both calcareous and fibrous components. The calcareous material is organized into columns that extend deep into the fibrous shell membrane. Many of the fibers of the membrane are enclosed within the crystalline matrix of the columns. Columns widen and flatten slightly at the outer surface of the eggshell to form cap-like structures composed of a compact crystalline matrix containing no fibers. The outer surface of eggs laid prior to completion of shell formation consists of a series of nodes obscured by a densely fibrous matrix. Similar nodes also are found at the inner surface of partially shelled eggs. The nodes represent the outer and inner aspects of columns that had not completed formation prior to oviposition. Our interpretation is that a layer (or layers) of the shell membrane forms first, with nucleation of columns occurring shortly thereafter. Columns grow into the membrane a short distance and enclose fibers of the membrane, but the primary direction of column growth is toward what will become the outer aspect of the shell. Calcareous columns and the shell membrane form more or less in concert until crystal growth outstrips that of the membrane and a cap-like apex of compact crystalline material is formed. The end result is an eggshell in which the shell membrane and calcareous material form a single unit for much of the thickness of the shell.     

Structure and properties of an under-sulfated heparan sulfate proteoglycan synthesized by a rat hepatoma cell line

A rat hepatoma cell line was shown to synthesize heparan sulfate and chondroitin sulfate proteoglycans. Unlike cultured hepatocytes, the hepatoma cells did not deposit these proteoglycans into an extracellular matrix, and most of the newly synthesized heparan sulfate proteoglycans were secreted into the culture medium. Heparan sulfate proteoglycans were also found associated with the cell surface. These proteoglycans could be solubilized by mild trypsin or detergent treatment of the cells but could not be displaced from the cells by incubation with heparin. The detergent-solubilized heparan sulfate proteoglycan had a hydrophobic segment that enabled it to bind to octyl- Sepharose. This segment could conceivably anchor the molecule in the lipid interior of the plasma membrane. The size of the hepatoma heparan sulfate proteoglycans was similar to that of proteoglycans isolated from rat liver microsomes or from primary cultures of rat hepatocytes. Ion-exchange chromatography on DEAE-Sephacel indicated that the hepatoma heparan sulfate proteoglycans had a lower average charge density than the rat liver heparan sulfate proteoglycans. The lower charge density of the hepatoma heparan sulfate can be largely attributed to a reduced number of N-sulfated glucosamine units in the polysaccharide chain compared with that of rat liver heparan sulfate. Hepatoma heparan sulfate proteoglycans purified from the culture medium had a considerably lower affinity for fibronectin-Sepharose compared with that of rat liver heparan sulfate proteoglycans. Furthermore, the hepatoma proteoglycan did not bind to the neoplastic cells, whereas heparan sulfate from normal rat liver bound to the hepatoma cells in a time-dependent reaction. The possible consequences of the reduced sulfation of the heparan sulfate proteoglycan produced by the hepatoma cells are discussed in terms of the postulated roles of heparan sulfate in the regulation of cell growth and extracellular matrix formation.     

Carbonic Anhydrase in Calcified Endoskeleton: Novel Activity in Biocalcification in Alcyonarian

Carbonic anhydrase (CA) is a key enzyme in the chemical reaction of living organisms and has been found to be associated with calcification in a number of invertebrates including calcareous sponges, but until now no direct evidence has been advanced to show CA activity in alcyonarian corals. However, it is essential to understand the role of CA in the process of biocalcification in alcyonarian. Here we describe the novel activity of CA and its relationship to the formation of calcified hard tissues in alcyonarian coral, Lobophytum crassum. We find that two CA proteins, which were partially purified by electro-elution treatment, can control the morphology of CaCO₃ crystals and one of them is potentially involved in the process of biocalcification. Previously, we isolated CA from the total extract of alcyonarian, and further, we report here a single protein, which has both calcium-binding and CA activities and is responsible for CaCO₃ nucleation and crystal growth. This matrix protein inhibited the precipitation of CaCO₃ from a saturated solution containing CaCl₂ and NaHCO₃, indicating that it can act as a negative regulator for calcification in the sclerites of alcyonarians. The effect of an inhibitor on the enzyme activity was also examined. These findings strongly support the idea that carbonic anhydrase domain in alcyonarian is involved in the calcification process. Our observations strongly suggest that the matrix protein in alcyonarian coral is not only a structural protein but also a catalyst.     

Effect of polyethylene glycol-400 at low concentrations on long-term growth of muscle phosphoglucomutase crystals from concentrated salt solutions.

Although rabbit muscle phosphoglucomutase occasionally deposits tetragonal crystals from solutions of ammonium sulfate at about 47% of saturation, low concentrations of polyethylene glycol-400 (PEG), 1 to 4.5% w/v, must be included to sustain crystal growth. A comparison of long-term growth rates for macroscopic crystals in the presence and absence of added PEG suggests that at high salt concentration this cosolute exerts its primary effect on disordered protein aggregates, either in the external medium or at the surface of the crystal, and thereby allows the growth of much larger crystals. Since the observed effects may arise from a PEG-induced increase in the "solubility" of the aggregate that exceeds the induced increase in solubility of the crystalline phase under these conditions, the physical basis for a cosolute-induced increase in solubility in the presence of a precipitant is considered. The applicability of such a rationale to the present system is supported by an assessment of the relative effects of polyethylene glycol and beta-octylglucoside on amorphous, salt-induced precipitates of phosphoglucomutase. PEG also produces what appears to be a differential effect on nucleation efficiency and crystal growth rate. Thus, seed crystals cannot be enlarged at a significant rate at high salt concentration without producing showers of extraneous nucleation centers when the concentration of added PEG is 3% or less. But PEG concentrations of 4.5% essentially eliminate the showering problem, ostensibly by increasing the supersaturation required for nucleation to a greater extent than that required for crystal growth. The same type of effect is observed during de novo growth. Again a solubility-based mechanism is posed. Hysteretic effects related to properties of amorphous aggregates of the protein also are described.     

Carbonate Precipitation of Bacterial Strains Isolated from Sediments and Seawater: Formation Mechanisms

This article presents a research study on carbonate formation in solid and liquid media by Thalassospira sp., Halomonas sp., Bacillus pumilus, and Pseudomonas grimontii, four bacterial strains isolated from sediments and deep seawater. As part of this study, we analyzed carbonic anhydrase activity, pH, adsorption of calcium and magnesium ions, and total organic and inorganic carbon. The geochemical program PHREEQC was also used to calculate the mineral saturation indexes in all the cultures. The minerals formed were studied with X-ray diffraction, X-ray dispersive energy microanalysis, and scanning electron microscopy. In addition, all four bacterial strains were found to induce carbonate precipitation and to have carbonic anhydrase activity. Sterile control experiments did not precipitate carbonate. In solid M1 and B4 media, all of the strains precipitated magnesium calcite, whereas in the liquid media, they precipitated different percentages of magnesium calcite, aragonite, and monohydrocalcite. In both cases, small amounts of amorphous precipitates were also produced. This article discusses carbonate formation and the possible role played by metabolic activity, bacterial surfaces and carbonic anhydrase in this process. Finally, the results obtained lead to a hypothesis regarding the importance of carbonate precipitation for the survival of bacteria populations in certain habitats.     

Protein purification by bulk crystallization: the recovery of ovalbumin

Crystallization is used industrially for the recovery and purification of many inorganic and organic materials. However, very little is reported on the application of bulk crystallization for proteins. In this work, ovalbumin was selected as a model protein to investigate the feasibility of using bulk crystallization for the recovery and purification of proteins. A stirred 1-L seeded batch crystallizer was used to obtain the crystal growth kinetics of ovalbumin in ammonium sulfate solutions at 30 degrees C. The width of the metastable region, in which crystal growth can occur without any nucleation, is equivalent to a relative supersaturation of about 20. The bulk crystallizations were undertaken within this range (using initial relative supersaturations less than 10) and nucleation was not observed. The ovalbumin concentration in solution was measured by UV absorbance and checked by crystal content measurement. Crystal size distributions were measured both by using a Malvern Mastersizer and by counting crystals through a microscope. The crystal growth rate was found to have a second-order dependence upon the ovalbumin supersaturation. While there is no discernible effect of ammonium sulfate concentration at pH 4.90, there is a slight effect at higher pH values. Overall the effect of ammonium sulfate concentration is small compared to the effect of pH, for which there is a 10-fold increase in the growth rate constant, k(Gsigma) over the range pH 4.6-5.4. To demonstrate the degree of purification which can be achieved by bulk crystallization, ovalbumin was crystallized from a solution containing conalbumin (80,000 Da) and lysozyme (14, 600 Da). After one crystallization and a crystal wash, ovalbumin crystals were produced with a protein purity greater than 99%. No contamination by the other proteins was observed when using overloaded sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) stained with Coomassie blue stain and only trace amounts of lysozyme were observed using a silver stain. The presence of these other proteins in solution did not effect the crystal growth rate constant, k(Gsigma). The study demonstrates the feasibility of using bulk crystallization for the recovery and purification of ovalbumin. It should be readily applicable to other protein systems. (c) 1995 John Wiley & Sons, Inc.     

Salt-Induced Dissociation of Carbonic Anhydrase from Intact Cells of Chlamydomonas reinhardtii

The extracellular carbonic anhydrase of Chlamydomonas reinhardtii is dissociated from either intact or lysed cells by treatment with a 20 millimolar potassium phosphate buffer containing 0.4 molar KCI at pH 7.4. Electrophoretic analysis of proteins dissociated by the high salt treatment reveals that carbonic anhydrase comprises over 70% of the total released. These results suggest that the extracellular carbonic anhydrase in C. reinhardtii is bound to either the cell wall or plasma membrane through ionic interactions.     

Influencing factors and formation mechanism of CaCO3 precipitation induced by microbial carbonic anhydrase

Microbial carbonic anhydrase promotes carbonate deposition, which is important in the formation and evolution of global carbon cycle and geological processes. A kind of bacteria producing extracellular carbonic anhydrase was selected to study the effects of temperature, pH value and Ca²⁺ concentration on bacterial growth, carbonic anhydrase activity and calcification rate in this paper. The results showed that the activity of carbonic anhydrase at 30 °C was the highest, which was beneficial to the calcification reaction, calcification rate of CaCO₃ was the fastest in alkaline environment with the initial pH value of 9.0. When the Ca2+ concentration was 60 mM, compared with other Ca²⁺ concentration, CA bacteria could grow and reproduce best, and the activity of bacteria was the highest, too low Ca²⁺ concentration would affect the generation of CaCO₃, while too high Ca²⁺ concentration would seriously affect the growth of bacteria and reduce the calcification rate. Finally, the mechanism of CaCO₃ precipitation induced by microbial carbonic anhydrase was studied. Carbonic anhydrase can accelerate the hydration of CO₂ into HCO₃⁻, and react with OH⁻ and Ca²⁺ to form CaCO₃ precipitation in alkaline environment and in the presence of calcium source.     

Acid-base disequilibrium in the venous blood of rainbow trout (Oncorhynchus mykiss)

Acid-base equilibria/disequilibria were evaluated in vivo in post-branchial arterial blood and pre-branchial venous blood of freshwater rainbow trout (Oncorhynchus mykiss). This was accomplished using arterial and venous extracorporeal circuits in conjunction with a stopped-flow apparatus. After the abrupt stoppage of circulating post-branchial blood within the stopped-flow apparatus, pH increased slowly ([Delta]pH = +0.032 ± 0.004 pH units; n = 15), thus confirming the existence of an acid-base disequilibrium state in the arterial blood of rainbow trout. The slow downstream pH changes were unaffected by prior treatment of fish with the carbonic anhydrase inhibitor benzolamide (1.2 mg kg^-1; [Delta]pH = +0.032 ± 0.01 pH units; n = 5) but were eliminated after intra-vascular injection of 10 mg kg^-1 bovine carbonic anhydrase ([Delta]pH = -0.011 ± 0.003 pH units; n = 8). These results demonstrate that the acid-base disequilibrium in the arterial blood reflects a total absence of extracellular carbonic anhydrase activity. Similar stopped-flow experiments revealed the existence of a reduced, yet significant, acid-base disequilibrium in the venous blood circulating within the caudal vein ([Delta]pH = +0.004 ± 0.003 pH units; n = 15). Selective inhibition of extracellular carbonic anhydrase using benzolamide did not significantly influence the magnitude of the venous pH disequilibrium ([Delta]pH = +0.007 ± 0.007 pH units; n = 8) whereas intra-vascular injection of carbonic anhydrase eliminated the pH disequilibrium. These results demonstrate that extracellular carbonic anhydrase, although reported to be present within the skeletal muscle of rainbow trout, does not accelerate post-capillary pH changes in the venous circulation.