The thin outer shell layer is composed of calcitic prisms, while the thick inner layer is built by aragonitic nacreous tablets. While the prismatic layer is black, the nacreous layer is black at the first stage, then becomes white (Figure 1a). The first stage of the nacreous layer is an organic deposit on the prisms, mainly on the thick organic walls surrounding the prismatic units (Figure 1b). Mineralized nacreous tablets follow a three-stage pattern (Figure 1c–e) from an irregular to a linear drawing. At last, in the white part of the inner surface, the growth pattern shows the classical spiral arrangement (Figure 1f). The nacreous tablets are arranged in regular layers (Figure 1g) described as “bricks and mortar”, because tablets are separated by organic lamellae. However, tablets are not pure mineral: they are a mixture of mineral and organic materials, as shown by fixed and etched samples (Figure 1h–j). A polished surface etched with the Mutvei’s solution (acetic acid + glutaraldehyde [21]) shows that tablets comprise parallel acicular crystallites parallel to the surface of the tablets, while remains of the interlamellar membranes are visible (Figure 1h). When the organic components are destroyed by commercial NaClO for 20 min, before using the Mutvei’s solution, another arrangement is revealed: parallel acicular crystallites are visible, perpendicular to the surface of the tablets. However, the surface shows that these crystallites are aligned, and are composed of small granules. An enzymatic etching (trypsine) shows that a tablet is composed of two sectors, the solubility of which differs (Figure 1j). Sectors are built by acicular crystallites. Such sectors are not revealed with another enzymatic hydrolysis (pronase), but the granular surface of the tablets is visible, the outer edge being less etched (Figure 1k). The heterogenous composition of a tablet and the different composition of the central part are also shown (Figure 1l).

Unetched and etched surfaces evidence the complexity of the structure and composition of the nacreous layer. Moreover, despite the fact that used enzymes are not specific for a protein, the resulting images displayed various aspects. Thus it may be inferred that the organic components in the interlamellar membranes and within the tablets are numerous.

Atomic force microscope (AFM) images also show the overlapping tablets, the surrounding organic membranes and the inner acicular crystallites (Figure 2a,b). Acicular crystallites are built of aligned granules (Figure 2c). These granules are surrounded by a thin cortex, as shown by phase image. Changes in color within granules suggest their heterogeneous composition (Figure 2d).

In an electrophoretic analysis, the pI of a component equals the pH of its zero mobility. In electrophoretic titration curves (ETC), this corresponds to the pH at which the component curve crosses the trough. Electrophoretic titration curves of bovine serum albumin (BSA) display two pI (4.6 and 5.6) (Figure 3a, white lines). Soluble nacreous matrix shows different electrophoretic mobilities at different pH values (Figure 3a). Coomassie blue stain of the nacreous components is more intense in the basic part of the gel, where the greatest difference in mobilities occurs. Discontinuities in the blue color confirm the heterogeneity and complexity of the nacreous matrix, the pI of which is acidic.

IPG strips are stained in 2D electrophoresis, showing that a part of the soluble matrix has high molecular weights (between 250 and 500 kDa) and does not penetrate into the second gel (Figure 3B, D). Using the usual fixative procedure, the immobilized pH gradient strip (IPG strip) is heavily stained in the acidic part (Figure 3b). The acidic part of the 2D gel is heavily stained, with discrete thin lines according to the pl, but with large smears according to the molecular weights. Some discrete faint bands are visible in the basic part of the gel. In the acidic—low molecular weights of the gel, a specific zone with no staining and a “cauliflower” pattern is present. Due to the Coomassie blue stain, the background of the Zn stain gel is blue. However, the discrete thin lines according to the pI, the “cauliflower pattern” and the smears in molecular weights are visible (Figure 3c). The patterns in the acidic parts of the Ag and Zn stained gels are similar. However, pI bands are more visible in the Zn stained gel. The second Ag stained gel, with the special fixative procedure [20], has a slightly different pattern (Figure 3d). IPG strip is stained also in the acidic part. In the second dimension gel, discrete bands are visible according to the pI, while only smears are visible for molecular weights. However, the “cauliflower” pattern of the gel 3b is absent, and a stained zone is shown in the basic—high molecular weights. More acidic components are stained on the right part of the gel. In this gel, a small band in the middle part of the pI range is not visible, as well as the basic part stained in Figure 3b.

HPLC profiles of the soluble matrix pI fractions vary from 1,500 kDa to 1 kDa (Figure 4a–j). The main peak is at 14 kDa (8–17 kDa range) in all the fractions, the second one being at about 42 kDa at 226 nm. However, ratios between the two peaks differ all along the pI range. Small peaks at about 1–2 kDa are also present. At 254 and 280 nm, the main peak is at about 42 kDa (Figure 4a–j). Some fractions show several strong peaks at 226 nm: the first acidic (pI 2.8–3.4, Figure 4a), the last basic (pI 9.59–10.07, Figure 4j), and an intermediate one (pI 5.8–6.15, Figure 4d). However, the main peak at 254 and 280 nm is also at about 42 kDa in these three fractions.

The main peak of the bulk soluble matrix is at about 103 kDa (Figure 4k), the second peak is at about 11 kDa at 226 nm. At 254 and 280 nm, the profiles are similar. An intermediate third peak is well marked at 254 and 280 nm, but appears as a shoulder at 226 nm. A weak shoulder is also present at about 1,500 kDa.

Despite the fact that nacre is the best-known layer, few components are separated and identified. Among them, most are proteins. Sugars and lipids are also present [22,23], but they are not well known. Proteins extracted from the insoluble and soluble matrix have been identified in the nacreous layer of several Pinctada species. Because of the diversity of preparative extraction, data are confused. Moreover, soluble matrices have been extracted from the insoluble matrix using different etching processes such as alkali solution [24]. Such results confirm the TEM observations of Nakahara [13] on the structure of the interlamellar membranes of the nacreous structure.

Despite some proteins being named from their apparent molecular weights, they often comprise several variants, so that the small differences are visible as irregularities in HPLC spectra or electrophoreses with a wide range of molecular weights. Bédouet et al. [25] have observed several bands in the acetic-soluble matrix of P. maxima: a main band at 20 kDa, and several faint bands at 60, 15, 12 and 9 kDa. Pereira-Mouries et al. [26] have confirmed that the concept of insoluble and soluble matrices depends on the demineralizing procedures. Both water-soluble and EDTA-soluble matrices appear as smears in SDS PAGE electrophoresis. However, two faint bands are visible at about 14 and 20 kDa in the EDTA-soluble matrix. They suggested that the 14 kDa band is similar to the acidic N14 protein (pI 4.8) described by Kono et al. [27] in P. maxima. P14 is known in the acetic acid-soluble matrix [28]. The acetic-soluble matrix of P. fucata yields the P60 protein, a complex composed of several units [29]. The nacrein, N28 and N35 proteins are included in the P60. The EDTA-soluble matrix of P. fucata was also analyzed [30,31]. Smears were observed between 45–60 kDa, and below 43 kDa. The EDTA-soluble matrix band at 55 kDa (nacrein) was shown to be glycosylated and sulfated [32]. Faint bands at 35, 25 and 21 kDa were also detected in P. fucata.

Attempts to solubilize the insoluble acidic extracts were more or less successful and provide additional data. It must be noted that in most cases, the used electrophoretic gels allow only “low” molecular weights (

An exact correlation between the results obtained by different methods is not easy. However, the main features are similar: low sugar content, low organic sulfate content, numerous proteins, and moderate to high pI.

Adult specimens of Pinctada margaritifera (Linnaeus, 1758) (Pteriomorpha, Pterioida, Pteriacea, Pteriidae) were collected in farms from French Polynesia. The shell is large, almost equivalve, with moderately convex valves. Concentric growth scales are present on the outer side of the valves. The outline is almost circular, and the outer color is greyish green. The inner part of the shell is white with black margins (Figure 1a).

Precision Plus standards (10–250 kDa), kaleidoscope pre-stained standards (6.9–202 kDa), gel filtration standards and ampholytes were from BioRad. Chondroitin sulfate A and trypsine were obtained from Sigma. Pronase was purchased from Merck.

To remove contaminants and residual tissues, samples were immersed in 3% NaClO for 1 hour. The samples were then rinsed with Milli-Q water, dried, and ground into powder. Powdered samples were immersed in 5 mL of Milli-Q water, then decalcified by progressive addition of 50% acetic acid to maintain the pH above 4. The entire extract was centrifuged at 20,000 g for 15 min to yield a soluble (SOM) and an insoluble (IOM) fraction. The soluble fraction was desalted by exchange with Milli-Q water on a micro-concentrator (Filtron) using a 3-kDa cut-off membrane and lyophilized.

Fractured and polished surfaces were used. Polished sections were etched with various acids and enzymes to reveal microstructural features; details of sample preparations are given in the figure legends. SEM observations were conducted using a Philips SEM 505 and XL30 microscopes. AFM observations were obtained with a Veeco Nanoscope III Dimension 3100. Samples were imaged at room temperature and in air. The probe consisted of a cantilever with an integrated silicon nitride tip. Micron scale images were acquired using tapping mode and phase imaging.

Electrophoretic titration curves (ETC): Non-denaturing electrophoresis was done to estimate the acidity of the soluble organic matrices. The charge intensity curve of proteins was determined with carrier ampholyte gels. Homogeneous polyacrylamide gels (T 4.2%, C 3%) with 3% ampholytes BioLyte pH 3–10, were prepared [33]. Components with molecular weights near 500 kDa can be detected with such gels. Following the electrophoretic run, the gels were placed in a fixing and staining colloidal Coomassie solution. Because of the absence of SDS in the gel and buffer, a washing phase is not necessary. Gels were then washed in water for 1h to remove the acid.

2D gel electrophoresis was done using the procedure described in Dauphin [34,35]. After electrophoresis and before Ag staining, two fixative procedures were used on two other gels: (1) the gel was placed in 40% methanol with 10% acetic acid for 24 h to fix the proteins and to remove the SDS; (2) the gel was fixed according to Gotliv et al. [20].

Preparative isoelectric focusing (IEF) was performed on a Rotofor cell (Biorad). A 5 W pre-run was done to allow the system to come to thermal equilibrium, so that the chiller was adjusted at 5 °C. The sample was mixed with ampholytes (pH 3–10) and Milli-Q water. Electrolyte buffers were: (1) anode NaOH 0.1M and cathode: H3PO4 0.1M. The power supply was set to 15 W constant power and the run was stopped when the voltage reached a constant value (total time ~4 h). Twenty fractions were obtained, fraction 1 at the anode pole and fraction 20 at the cathode pole. After focusing, the solution in each compartment is collected. Ampholytes were removed using 3 kDa Microsep centrifugated for 75 min at 5,000 g; then, purified samples were lyophilized.

Chromatographic analyses were performed using two TSK G5000PW and G3000PW columns. These columns were used in series allowing the separation of proteins from 200 to 1 × 106 Da. They were eluted with 0.2 mM Tris, pH 7.5 at a flow rate of 0.75 mL/min using a high sensitivity refractive index detector PE 200 (Perkin-Elmer), and a multiple wavelength—diode array detector Agilent series1200 and monitored at 210, 226, 254 and 278 nm. The quantity of soluble matrices in each of the 20 fractions resulting from IEF was low. Thus, two successive fractions were mixed to obtain 10 fractions. The lyophilized SOM was dissolved in the buffer overnight, and all analyses were performed at ambient temperature.