136
Universidad de Sonora
ISSN: 1665-1456
136
Journal of biological and health sciences
http://biotecnia.unison.mx
Volume XXV, Issue 2 *Author for correspondence: Norma Yolanda Hernandez Saavedra
e-mail: nhernan04@cibnor.mx
Received: October 31, 2022
Accepted: January 23, 2023
Eect of ion and protein concentration of Ps19, a shell protein from
Pteria sterna, on calcium carbonate polymorph
Efecto de iones y concentración de proteína Ps19, una proteína de la concha de Pteria sterna,
en los polimorfos de carbonato de calcio
Raquel Gabriela Arroyo-Loranca1, Crisalejandra Rivera-Perez2, Luis Hernandez-Adame2, Ariel Arturo Cruz Villacorta1,
Jose Luis Rodriguez-Lopez3, Norma Yolanda Hernandez-Saavedra1*
1 Molecular Genetics Laboratory, Center for Biological Research of the Northwest (CIBNOR), La Paz, Baja California South,
Mexico.
2 CONACYT. Biological Research Center of the Northwest (CIBNOR), La Paz, Baja California Sur, Mexico.
3 Advanced Materials Department, Potosi Institute for Scientific and Technological Research, San Luis Potosi, Mexico.
ABSTRACT
Calcium carbonate is present in many biological structures
such as bivalve shells, which is composed mainly of two
CaCO3 polymorphs: calcite and aragonite. However, other
forms of calcium carbonate exist like vaterite and amorphous
calcium carbonate (ACC) that are not commonly reported.
Polymorph selection is inuenced by salt concentration, co-
factor ions, and the presence of shell matrix proteins (SMPs)
which regulates calcium carbonate deposition, among other
factors. In this study, in vitro calcium carbonate crystallization
of four dierent saline solutions (1: 40 mM CaCl2, MgCl2,
100 mM NaHCO3; 2: CaCl2, 100 mM Na2CO3; 3: 40 mM CaCl2,
MgSO4, 100 mM Na2CO3; 4: CaCl2/MgCl2, 100 mM NaHCO3) at
two molarities (40 o 100 mM) was evaluated with increased
concentrations of the Ps19 protein (0.2, 0.7 y 1.2 µg/µL), an
insoluble extracted protein from the Pteria sterna shell, pre-
viously described as a promotor of aragonite platelet crystalli-
zation. In vitro crystallizations showed that Ps19 is capable to
induce aragonite and calcite deposition in a dose-dependent
manner, but also vaterite under certain conditions, acting
as a promoter and inhibitor of crystallization. The results
contribute to understand how Ps19 control precipitation of
calcium polymorphs in the growth of the prismatic and nacre
layer of the shell of P. sterna.
Keywords: Mollusk, shell protein, calcium carbonate, crysta-
llization, nacre.
RESUMEN
El carbonato de calcio está presente en muchas estructuras
biológicas, como la concha de bivalvo, que se compone prin-
cipalmente de dos polimorfos de CaCO3: calcita y aragonito.
Sin embargo, existen otras formas de carbonato de calcio
como vaterita y carbonato de calcio amorfo (ACC) que no
se reportan comúnmente. La selección de polimorfos está
inuenciada por la concentración de sal, los iones cofactores
y la presencia de proteínas de la matriz de la cubierta (SMP)
que regulan la deposición de carbonato de calcio, entre otros
factores. En este estudio, se evaluó la cristalización in vitro de
carbonato de calcio de cuatro soluciones salinas diferentes
(1: 40 mM CaCl2, MgCl2, 100 mM NaHCO3; 2: CaCl2, 100 mM
Na2CO3; 3: 40 mM CaCl2, MgSO4, 100 mM Na2CO3; 4: CaCl2/
MgCl2, 100 mM NaHCO3) en dos molaridades (40 o 100 mM)
con diferentes concentraciones la proteína Ps19 (0.2, 0.7 y
1.2 µg/µL) una proteína insoluble extraída de la concha de
Pteria sterna, descrita anteriormente como promotora de la
cristalización de plaquetas de aragonita. Las cristalizaciones
in vitro mostraron que Ps19 es capaz de inducir la deposición
de aragonita y calcita de forma dependiente de la dosis,
pero también de vaterita en determinadas condiciones, ac-
tuando como promotor e inhibidor de la cristalización. Los
resultados contribuyen a comprender cómo Ps19 controla la
precipitación de polimorfos de calcio en el crecimiento de la
capa prismática y de nácar de la concha de P. sterna.
Palabras clave: Molusco, proteína de la concha, carbonato,
cristalización, nácar.
INTRODUCTION
In nature, three dierent anhydrous crystalline polymorphs
of calcium carbonate (CaCO3) exist (calcite, aragonite, and
vaterite), two well-dened hydrous crystalline polymorphs
(calcium carbonate monohydrate and calcium carbonate
hexahydrate), and one amorphous form (ACC) (Meldrum and
Colfen, 2008). The biological process by which CaCO3 poly-
morphs are synthesized is called biomineralization (Kocot et
al., 2016; Song et al., 2019). The shell of mollusks is the most
studied CaCO3 biomineral, because is the most abundant
biomineral in nature and it is relatively easy to obtain (Demi-
chelis et al., 2018). The shell of bivalves is mainly composed
of CaCO3 (95 - 99 %) and an organic matrix containing acidic
proteins, ꞵ-chitin, glycoproteins among other molecules (1
- 5 %) that function as a scaold for mineral nucleation and
plate formation (Wolf et al., 2013).
The periostracum, the outer layer of the shell, is the rst
defense of the organism against external agents, it is not
calcied and it is made of organic compounds (Kocot et al.,
2016). After the periostracum, the prismatic layer is compo-
sed mainly of calcite, the most stable and the second most
abundant CaCO3 polymorph in mollusks; it crystallizes in a
DOI:10.18633/biotecnia.v25i2.1885
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rhombohedral system making it a resistant and tough mate-
rial to penetrate by forming prismatic structures (Bahn et al.,
2017). Beneath prismatic layer is the mother of pearl (nacre)
layer, which is formed by aragonite. This polymorph has an
orthorhombic form and it is less stable than calcite in isolated
conditions, however, in the shell becomes a tougher, stier,
and stronger material by assembling aragonite plates into
structures that resemble a “brick-wall” (Evans, 2019). Besides
calcite and aragonite, mollusks are capable to manipulate
vaterite, the most unstable CaCO3 polymorph present in the
shell when their shell suers a deformation (Wilt, 2005).
Vesicles of the mantle in mollusks act as ion storage sites
of vaterite or amorphous calcium carbonate (ACC) (Addadi et
al., 2006). ACC is the hydrated form of CaCO3 with a poorly or-
der that contains magnesium (Addadi et al., 2003), and acts a
transient precursor of more stable CaCO3 polymorphs (Politi
et al., 2008). There are two models that try to explain CaCO3
nucleation. The rst model, initiates with prenucleation, in
which the ionic solution creates metastable clusters to begin
the nucleation phase were single ions attach to the cluster
and nally, depending on thermodynamic factors, calcite,
vaterite, aragonite or ACC will be formed in the postnuclea-
tion phase. In second model, the prenucleation phase is
pH-dependent to form a stable cluster, where the nucleation
phase will begin depending on cluster concentration, its
aggregation, or ion attachment. During the postnucleation
phase, ACC is formed, and then calcite, aragonite, and vateri-
te are crystallized (Demichelis et al., 2018).
Shell growth in mollusk is mediated by the shell matrix
proteins (SMPs) from the mollusk shells. SMPs are synthesi-
zed in the mantle cells and released to the extrapallial uid
(EPF), between the mantle and the inner face of the shell
(Wilt, 2005). The EPF contains a variety of ions which interact
with SMPs (Wilt, 2005). SMPs are classied as soluble and
insoluble proteins according to their solubility after the de-
calcication procedure used to extract them from the shell.
Soluble proteins are rich in acidic hydrophilic residues, they
are found between the crystals forming part of the scaol-
ding where CaCO3 deposits (Levi-Kalisman et al., 2001), while
insoluble proteins are found inside the crystals, they have a
high proportion of aliphatic amino acids forming short repe-
titive domains that have been associated with nucleation by
promoting the interaction with acidic polyanionic soluble
proteins (Wolf et al., 2013; Du et al., 2018).
Crystal polymorph growth in the mollusk shell is mo-
dulated by SMPs protein concentration and by the presence
of cofactors. Low protein concentration (1 µg/mL) of some
SMPs (e.g. PfN44), have been related to calcite growth with
few aragonite crystals formation, but the increase in protein
concentrations (2.5 µg/mL) have shown a total inhibition of
aragonite deposition (Pan et al., 2014). However, other SMPs
such as Pif97 and pearlin showed inhibition of calcite crystal
growth and ACC stabilization in vitro in increasing amounts
of proteins (Montagnani et al., 2011; Bahn et al., 2015). This
signicant discrepancy between proteins could be related
to their function in shell formation, either as a modulator of
calcite or aragonite, or both. Besides protein concentration,
Mg2+ ions have been reported to modulate aragonite growth
(Pan et al., 2014; Ma and Feng, 2015). Low magnesium con-
tent induces calcite growth while high magnesium content
induces the formation of aragonite (Raz et al., 2000). In
Hyriopsis cumingii, the water-soluble matrix of aragonite
pearls formed little needle-like crystals, accompanied by irre-
gular Mg-calcite structures when low magnesium is present
(10 mM). However, in the presence of high magnesium ions
(40 mM), quasi-spherical aragonite aggregates appear from
needle-like crystals, coexisting with a few Mg-calcites (Ma
and Feng, 2015).
Other proteins, such as SPARC (secreted protein acidic
and rich in cysteine), from P. fucata, participate in nacre
formation by stabilizing vaterite to inhibit calcite, as well as
by forming aragonite in presence of Mg2+ or other proteins
(Xie et al., 2016). Nevertheless, there are also proteins able
to produce aragonite crystals in presence of magnesium or
calcium (40 mM) as cofactors, such as the glycoprotein Ps19
from Pteria sterna (Arroyo-Loranca et al., 2020). Even when
this information suggests that SMPs can to modulate crys-
tal polymorphs by protein and ion concentrations, further
research is needed to support this statement since the crys-
tallization assay previously reported in several SMPs are not
always comparable due to the dierent preparations used.
To understand the biomineralization processes is ne-
cessary to known the mechanism used by SMPs to control
crystal deposition, either by protein and/or ion concen-
tration. Therefore, this research aimed to understand the
role of Ps19, a novel SMPs, with no homology to previously
described proteins, on crystal deposition, by evaluating the
eect of protein concentration (0.2 – 1.2 μg·μL-1) and cofactor
concentration (40/100 mM of MgCl2, CaCl2, 1:1 MgCl2:CaCl2
and MgSO4) on crystal polymorph deposition in vitro. The re-
sults indicate Ps19 is an eective promoter of aragonite and
calcite in the presence of MgCl2 and CaCl2, respectively. Also,
this positive modulation is dependent on protein and ion
concentration. Thus, this suggests that Ps19 may be involved
in the prismatic and nacre layer of Pteria sterna.
MATERIAL AND METHODS
Ps19 extraction from P. sterna shell
Perlas del Cortez S. de R.L. MI., granted three adult organisms
from Bahia de La Paz, B.C.S. Oysters were transported to the
Centro de Investigaciones Biológicas del Noroeste S.C. faci-
lities where shells were pulverized, and 20 g of the powder
were decalcied with cold acetic acid (10 % v/v) at 4 °C and
constant stirring according to Montagnani et al. (2011); later,
the solution was centrifuged to obtain the Acetic Soluble Ma-
trix (ASM) and the Acetic Insoluble Matrix (AIM) as described
by Arroyo-Loranca et al. (2020).
SDS-PAGE
The AIM, and later the Ps19 protein were loaded into a sodium
dodecyl sulphate polyacrylamide gel electrophoresis (SDS-
PAGE) according to Laemmli (1970), the samples were trea-
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Arroyo-Loranca et al: Biotecnia / XXV (2): 136-145 (2023)
138
ted as previously described by Arroyo-Loranca et al. (2020). A
quantity of 60.3 μg of AIM and 15.1 μg of P. sterna shell Ps19
per gel were loaded. An SDS-PAGE molecular weight marker
was used for molecular weight comparison (Broad range,
Bio-Rad 161-0317). Electrophoresis was conducted at 90-V
at room temperature, using a Bio-Rad electrophoresis unit
(Protean II). After electrophoresis, the gel was stained with
Coomassie Brilliant Blue R250 (CBB) for 2 h, washed out, and
analyzed using a Chemi Doc XRS (Bio-Rad).
Protein quantication by densitometry
Ps19 quantication was performed by pixel densitometry in
a 16 % SDS-PAGE gel stained with CBB. A protein standard
curve was constructed with ovalbumin protein (0.25 - 8.0
μg·μL-1) as described by Arroyo-Loranca et al. (2020). A Chemi
Doc XRS (Bio-Rad, California, USA) was used to analyze the
image of the gel to obtain the linear equation (1) and calcula-
te the quantity of the proteins present in the samples.
(1)
The Ps19 protein was puried from the AIM of P. sterna
shell by four preparative polyacrylamide gel electrophoresis
according to the Mini-Prep Cell manual (Bio-Rad, 491 Prep
Cell) according to Arroyo-Loranca et al. (2020). A total of 100
fractions of 200 μL each were collected, selected fractions
were loaded into an SDS-PAGE polyacrylamide gel (16 %)
to identify the fractions containing the Ps19 protein. Then,
fractions containing Ps19 were pooled, concentrated, and
cleaned from the electrophoresis buer by centrifugation
with an Amicon Ultra-4 lter (EMD Millipore) as described by
Arroyo-Loranca et al. (2020). The puried protein (380 μL) was
stored at -20 °C.
Ps19 identication
The puried Ps19 was analyzed to verify the properties of
the previously characterized protein by Arroyo-Loranca et al.
(2020). The molecular weight of Ps19 was calculated through
its Rf, the calcium-binding capability was corroborated by
the Stains-All stain (Green et al., 1973) after electrophoresis
separation as previously described. A total of 15.1 μg of Ps19
in an SDS-PAGE 16 % polyacrylamide gel was used.
Calcium carbonate crystallization in vitro
Calcium carbonate (CaCO3) crystallization in the presence of
Ps19 was evaluated by the incubation of four saturated solu-
tions and three dierente protein concentrations that shown
in Table 1. Each combination (10 μL of Ps19 and 50 μL of satu-
rated solution) was placed over a glass coverslip inside a Petri
dish with absorbent paper at the bottom to prevent conden-
sation. The Petri dishes were sealed with paralm, placed into
a container, and incubated for 30 days at 4 °C. Controls and
solutions 1 to 4 (50 μL) were mixed with sterile distilled water
(10 μL) instead of Ps19 and the protein by itself (10 μL) was
mixed with 50 μL of sterile distilled water. The morphology of
the crystals was analyzed by Scanning Electron Microscopy
(SEM) at the Electronic Microscopy Laboratory at Centro de
Investigaciones Biológicas del Noroeste (CIBNOR), México.
Every experiment and analysis were performed by triplicate.
Raman spectroscopy
Raman spectra were identied by using an InVia micro-
Raman spectrometer (Renishaw, Gloucestershire, UK) with
an excitation line of 532 nm provided by a YAG laser of 100
mW and a spot size of 2 μm x 2 μm. For measurements, the
slits were set at 200 μm and a 100× objective was used. The
crystals were scanned by triplicate for 90 seconds from 100 to
1900 cm−1 for the specic identication.
RESULTS
Ps19 isolation and quantication
The acid-soluble (ASM) and acid-insoluble (AIM) matrixes
were obtained by extraction with cold acetic acid. The AIM
was separated by denaturalizing electrophoresis and visuali-
zed by CBB stain (Figure 1A). The Ps19 protein was identied
Table 1. Conditions for CaCO3 crystallization in vitro in the presence of Ps19.
Tabla 1. Condiciones para la cristalización de CaCO3 in vitro en presencia de Ps19.
Solution SaltCofactor
Molarity of the
cofactor
(Mm)
pH Ps19 (μg·μL-1)Expected CaCO3
polymorph Reference
140 mM CaCl2,
100 mM NaHCO3 MgCl240 or 100 8.2
0.2
Aragonite (Weiss et al.,
2000)
0.7
1.2
2 100 mM Na2CO3 CaCl240 or 100 8.2
0.2
Calcite (Declet et al.,
2016)
0.7
1.2
340 mM CaCl2,
100 mM Na2CO3 MgSO440 or 100 8.2
0.2
Aragonite (Nielsen et al.,
2016)
0.7
1.2
4 100 mM NaHCO3 CaCl2 and MgCl240 or 100 8.2
0.2
Calcite/ aragonite (Loste et al.,
2003)
0.7
1.2
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by determining its molecular weight (~19 kDa) through its
relative mobility along with the gel.
The Ps19 protein was puried by preparative electro-
phoresis (Figure 1B). From the four electrophoreses, a total
of 576 μg of Ps19 were puried from 3.4 mg of unpuried
protein present in the AIM extracted from the pulverized
shell, having a protein (Ps19) with a 17 % yield and 94 %
purity (Table 2).
The amount of puried Ps19 was determined by pixel
densitometry (Figure 1B). The CBB and Stains-All stains co-
rroborated that the isolated protein was Ps19, which had the
expected molecular weight (19 kDa) and presented Ca2+ bin-
ding capability (Figure 1B) as described by Arroyo-Loranca et
al. (2020).
CaCO3 crystallization in vitro
The modulation of CaCO3 crystal polymorph formation by
Ps19 was evaluated by SEM. The Ps19 protein was able to
produce calcite crystals when MgCl2 was used as a cofactor
at 40 and 100 mM (Figure 2). At 40 mM MgCl2, Ps19 crysta-
llized calcite crystal plates in their typical geometric shape
with smooth sides (Figure 2 F-H), which were comparable
in size than those observed in the controls (Figure 2E, 3I-K).
At 100 mM MgCl2 (Figure 2), only hexagonal vaterite was
formed, plates were more structured, and stacked forming
platelet interlocks (white arrow, Figure 2B). Increase of Ps19
concentration displayed crystal inhibition at 40 mM when
protein was set at 1.2 µg·μL-1, however, this behavior was not
Figure 1. Acetic insoluble matrix (AIM) from the shell of Pteria sterna. (A) AIM in a SDS-PAGE 16
% polyacrylamide gel stained with CBB. MWS: molecular weight standard (Bio-Rad 1610317), AIM:
acetic-acid insoluble matrix proteins, the arrow indicates the Ps19 protein. (B) Isolated Ps19 shell
protein from P. sterna and Ca2+ binding capability (Stain all). Ova: ovalbumin (negative control).
Figura 1. Matriz acética insoluble (AIM) de la concha de Pteria sterna. (A) AIM en un gel de polia-
crilamida SDS-PAGE al 16 % teñido con CBB. MWS: estándar de peso molecular (Bio-Rad 1610317),
AIM: proteínas de matriz insolubles en ácido acético, la echa indica la proteína Ps19. (B) Proteína
de la concha Ps19 de P. sterna y capacidad de unión a calcio (tinción Stain all). Ova: Ovoalbúmina
(control negativo).
Table 2. Ps19 purication from Pteria sterna shell.
Tabla 2. Puricación de Ps19 a partir de la concha de Pteria sterna.
Species Step
Total
Protein
(mg)
Ps19
(mg)
Yield
(%)
Purity
(%)
Pteria
sterna Crude extract 3.456 3.016 100 87
Preparative
electrophoresis 3.016 0.616 20 94
Amicon-washed
concentrate 0.576 0.576 19 94
observed at 100 mM MgCl2, and CaCO3 crystals presented
diamond shape instead of hexagonal, as seen at 0.2 and 1.2
µg·µL-1 Ps19 concentration.
The addition of Ps19 to the solution containing 40 and
100 mM CaCl2 did not generate crystals of calcite or aragonite
(Figure 3). At 40 mM rod-like crystals were observed (Figure
3F, G), while high CaCl2 concentration displayed scarce rod-
like crystals (Figure 3B) and isolated polycrystalline deposits
generating halos or inhibition zones (Figure 3C). The increase
of Ps19 concentration at 40 and 100 mM did not produce any
signicant pattern on crystal deposition.
Saturated solution containing MgSO4 as source of Mg2+
ion resulted in CaCO3 precipitation as ACC (Figure 4). At 40
mM MgSO4, ACC polymorph was predominant in the solu-
tion, however small aragonite structures were visible at 0.2
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140
Figure 2. SEM micrographs of calcium carbonate crystals growth in vitro in the presence of 40 mM CaCl2, Mg2+ ion (40 and 100 mM), 100 mM NaH-
CO3 and Ps19 at increasing concentrations (µg·µL-1). First panel from left to right (A, E) Negative controls without Ps19; 100 mM Mg2+ ion in presence of
(B) 0.2 µg·µL-1, (C) 0.7 µg·µL-1, (D) 1.2 µg·µL-1 Ps19; 40 mM Mg2+ ion in presence of (B) 0.2 µg·µL-1, (C) 0.7 µg·µL-1, (D) 1.2 µg·µL-1 Ps19; (I-K) protein alone without
salt. Scale bars are 500 µm. Hexagonal structures are shown in white lines, and arrow indicates platelet interlocks. Each picture is representative of three
independent crystallization assays.
Figura 2. Micrografías SEM del crecimiento de cristales de carbonato de calcio in vitro en presencia de CaCl2 40 mM, iones Mg2+ (40 y 100 mM), Na-
HCO3 100 mM y Ps19 a concentraciones crecientes (µg·µL-1). Primer panel de izquierda a derecha (A, E) Controles negativos sin Ps19; ion Mg2+ 100 mM en
presencia de (B) 0,2 µg·µL-1, (C) 0,7 µg·µL-1, (D) 1,2 µg·µL-1 Ps19; ion Mg2+ 40 mM en presencia de (B) 0,2 µg·µL-1, (C) 0,7 µg·µL-1, (D) 1,2 µg·µL-1 Ps19; (I-K) proteí-
na sola sin sal. Las barras de escala son de 500 µm. Las estructuras hexagonales se muestran en líneas blancas y la echa indica interbloqueos de plaquetas.
Cada imagen es representativa de tres ensayos de cristalización independientes.
Figure 3. SEM micrographs of calcium carbonate crystals growth in vitro in the presence of Ca2+ ion (40 and 100 mM), 100 mM Na2CO3 and Ps19 at increasing concentrations
(µg·µL-1). First panel from left to right (A, E) Negative controls without Ps19; 100 mM Ca2+ ion in presence of Ps19 protein (B) 0.2 µg·µL-1, (C) 0.7 µg·µL-1, (D) 1.2 µg·µL-1 Ps19; 40 mM Ca2+ ion
in presence of (B) 0.2 µg·µL-1, (C) 0.7 µg·µL-1, (D) 1.2 µg·µL-1 Ps19; (I-K) protein alone without salt. Scale bars are 500 µm. Each picture is representative of three independent crystallization
assays.
Figura 3. Micrografías SEM del crecimiento in vitro de cristales de carbonato de calcio en presencia de ion Ca2+ (40 y 100 mM), Na2CO3 100 mM y Ps19 a concentraciones
crecientes (µg·µL-1). Primer panel de izquierda a derecha (A, E) Controles negativos sin Ps19; ion Ca2+ 100 mM en presencia de (B) 0,2 µg·µL-1, (C) 0,7 µg·µL-1, (D) 1,2
µg·µL-1 Ps19; ion Ca2+ 40 mM en presencia de (B) 0,2 µg·µL-1, (C) 0,7 µg·µL-1, (D) 1,2 µg·µL-1 Ps19; (I-K) proteína sola sin sal. Las barras de escala son de 500 µm. Cada
imagen es representativa de tres ensayos de cristalización independientes.
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and 0.7 µg·µL-1 Ps19 protein (Figure 4F-H). But, at 100 mM
MgSO4 no rhombohedral or even facetted crystals at all were
spotted, instead agglomerations of spheres were observed
at all Ps19 concentrations tested (Figure 4B-D).
Finally, when CaCl2/MgCl2 (1:1 ratio) were present in the
crystallization assay formed scarce crystal structures and
a thin lm was present over all crystal preparations (Figure
5). At 40 mM CaCl2/MgCl2, an aragonite tablet was observed
at 0.7 µg·µL-1 (Figure 5G), while at 100 mM CaCl2/MgCl2, only
aragonite crystals were observed at 0.7 µg·µL-1 and 1.2 µg·µL-1
(Figure 5C,D), which were 100 µm size.
Figure 4. SEM micrographs of calcium carbonate crystals growth in vitro in the presence of 40 mM CaCl2, Mg2+ ion (40 and 100 mM) as MgSO4, 100
mM Na2CO3 and Ps19 at increasing concentrations (µg·µL-1). First panel from left to right (A, E) Negative controls without Ps19; 100 mM Mg2+ ion in pre-
sence of (B) 0.2 µg·µL-1, (C) 0.7 µg·µL-1, (D) 1.2 µg·µL-1 Ps19; 40 mM Mg2+ ion in presence of (B) 0.2 µg·µL-1, (C) 0.7 µg·µL-1, (D) 1.2 µg·µL-1 Ps19; (I-K) protein alone
without salt. Scale bars are 500 µm, inlets are 50 µm size. Each picture is representative of three independent crystallization assays.
Figura 4. Micrografías SEM del crecimiento de cristales de carbonato de calcio in vitro en presencia de CaCl2 40 mM, iones Mg2+ (40 y 100 mM) como
MgSO4, Na2CO3 100 mM y Ps19 a concentraciones crecientes (µg·µL-1). Primer panel de izquierda a derecha (A, E) Controles negativos sin Ps19; ion Mg2+
100 mM en presencia de (B) 0.2 µg·µL-1, (C) 0.7 µg·µL-1, (D) 1.2 µg·µL-1 Ps19; ion Mg2+ 40 mM en presencia de (B) 0.2 µg·µL-1, (C) 0.7 µg·µL-1, (D) 1.2 µg·µL-1 Ps19;
(I-K) proteína sola sin sal. Las barras de escala son de 500 µm, las entradas tienen un tamaño de 50 µm. Cada imagen es representativa de tres ensayos de
cristalización independientes.
Figure 5. SEM micrographs of calcium carbonate crystals growth in vitro in the presence of CaCl2 and MgCl2 (40 and 100 mM), 100 mM NaHCO3 and
Ps19 at increasing concentrations (µg·µL-1). First panel from left to right (A, E) Negative controls without Ps19; 100 mM CaCl2/MgCl2 in presence of (B) 0.2
µg·µL-1, (C) 0.7 µg·µL-1, (D) 1.2 µg·µL-1 Ps19; 40 mM CaCl2/MgCl2 in presence of (B) 0.2 µg·µL-1, (C) 0.7 µg·µL-1, (D) 1.2 µg·µL-1 Ps19; (I-K) protein alone without salt.
Scale bars are 500 µm, inlets are 50 µm size. Each picture is representative of three independent crystallization assays.
Figura 5. Micrografías SEM del crecimiento in vitro de cristales de carbonato de calcio en presencia de CaCl2 y MgCl2 (40 y 100 mM), NaHCO3 100
mM y Ps19 a concentraciones crecientes (µg·µL-1). Primer panel de izquierda a derecha (A, E) Controles negativos sin Ps19; CaCl2/MgCl2 100 mM en presen-
cia de (B) 0.2 µg·µL-1, (C) 0.7 µg·µL-1, (D) 1.2 µg·µL-1 Ps19; CaCl2/MgCl2 40 mM en presencia de (B) 0.2 µg·µL-1, (C) 0.7 µg·µL-1, (D) 1.2 µg·µL-1 Ps19; (I-K) proteína
sola sin sal. Las barras de escala son de 500 µm, las entradas tienen un tamaño de 50 µm. Cada imagen es representativa de tres ensayos de cristalización
independientes.
142 Volume XXV, Issue 2
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142
Raman analysis
Raman measurements conrmed the presence of CaCO3
polymorphs (Figure 6). The spectra show characteristics
peaks belonging to the internal vibration mode of (at
around 1080 cm-1) and the related between Ca2+ with
(Han et al., 2017). The signal dierences from 100 to 400 cm-1
derived from the dierent vibration modes of Ca2+ and
in calcite and aragonite. In aragonite, the interatomic distan-
ces between Ca2+ and are smaller than that of calcite;
hence, the bonds between Ca2+ and are stronger and the
Raman signals appear at lower-frequency vibrational mode
than that of calcite (Arroyo-Loranca et al., 2020). Moreover, is
interesting to observe the presence of a triplet formed in the
region from 1070 to 1090 cm-1. These vibrations are charac-
teristic of the vaterite formation that was conrmed by the
additional vibrations modes from 700 to 790 cm-1 belonging
to the internal translational modes of in-plane bending of the
carbonate ions in vaterite (Soldati et al., 2008). In this sense,
Figure 6 shows the Raman spectra of CaCO3 polymorphs
when MgCl2 was used as a cofactor at 40 and 100 mM. At 40
mM, the control (solution without protein) shows a triplet
from 1070 to 1090 cm-1 assigned to the vaterite formation,
while a protein concentration of 0.2 and 0.7 µg·μL-1 show
vibration modes in the range of 700 and 200 cm-1 assigned to
the calcite formation.
The Raman spectra of samples using CaCl2 as a cofactor
are shown in Figure 7. Here we observe a single vibration
mode in the region from 1060 to 1080 cm-1corresponding
to which indicates only a deposition without any
structured crystals formation as can be observed by SEM (Fi-
gure 3). Figure 8 shows the Raman spectra of samples using
MgSO4 as a cofactor. The spectra show vibration modes in
the region from 900 to 1070 cm-1 and from 230 to 640 cm-1.
These vibrations modes can be assigned to ACC polymorphs.
Finally, the use of CaCl2 and MgCl2 as a cofactor, do not show
any crystal formation.
DISCUSSION
The shell growth in Mollusk has been described to start
with the precipitation of ACC, which is known to surround
aragonite platelets in gastropods (e.g. Haliotis laevigata) and
bivalves (e.g. Pinctada margaritifera and Atrina rigida) indica-
Figure 6. Raman spectra of calcite and vaterite formation by using
Ps19 protein and MgCl2 as a cofactor. Calcium carbonate crystals
growth in vitro in the presence of MgCl2 Na2CO3. (A) 100 mM and (B)
40 mM, respectively. Color line indicate salts preparations, red line: salt
without protein added, black: salt in the presente of 0.2 µg·µL-1, blue: salt in
the presence of 0.7 µg·µL-1, brown: salt in the presence of 1.2 µg·µL-1. Color
arrows indicate the peaks for each salt preparation.
Figura 6. Espectros Raman de formación de calcita y vaterita usando
proteína Ps19 y MgCl2 como cofactor. Crecimiento de cristales de car-
bonato de calcio in vitro en presencia de MgCl2 Na2CO3. (A) 100 mM y
(B) 40 mM, respectivamente. La línea de color indica preparaciones de sales,
línea roja: sal sin proteína añadida, negra: sal en presencia de 0.2 µg·µL-1,
azul: sal en presencia de 0.7 µg·µL-1, marrón: sal en presencia de 1.2 µg·µL-1.
Las echas de color indican los picos para cada preparación de sal.
Figure 7. Raman spectra of carbonates deposition by using Ps19
protein and CaCl2 as a cofactor. Calcium carbonate crystals growth in vitro
in the presente of CaCl2 NaHCO3 (A) 100 mM and (B) 40 mM, respectively.
Color line indicate salts preparations, red line: salt without protein added,
black: salt in the presente of 0.2 µg·µL-1, blue: salt in the presence of 0.7
µg·µL-1, brown: salt in the presence of 1.2 µg·µL-1. Color arrows indicate the
peaks for each salt preparation.
Figura 7. Espectros Raman de deposición de carbonatos usando pro-
teína Ps19 y CaCl2 como cofactor. Crecimiento de cristales de carbonato
de calcio in vitro en presencia de CaCl2 NaHCO3 (A) 100 mM y (B) 40 mM,
respectivamente. La línea de color indica preparaciones de sales, línea roja:
sal sin proteína añadida, negra: sal en presencia de 0.2 µg·µL-1, azul: sal en
presencia de 0.7 µg·µL-1, marrón: sal en presencia de 1.2 µg·µL-1. Las echas
de color indican los picos para cada preparación de sal.
143
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143
ting a nascent growing platelet (Nassif et al., 2005; Rousseau
et al., 2009). ACC is a highly metastable phase, which is often
found hydrated, and after dehydration, transforms to calcite,
aragonite, or vaterite (Radha et al., 2010). Divalent ions have
been found to aect the crystallization rates and pathways
of ACC (Tobler et al., 2015). In Mollusk shells, Mg2+ ion is 2.55
% of their weight (Huang et al., 2018). This ion is known to
stabilize ACC forming a complex (Mg-ACC) and reducing
their reactivity to form polymorphs or calcite or aragonite,
however, aragonite can be detectable since Mg2+ is known to
suppress calcite growth (Davis et al., 2000). In our study, the
mixture of the Ps19 with the saturated solution containing 40
and 100 mM MgSO4, respectively (Figure 4), resulted in the
formation of imperfect spherical morphologies, which has
been previously described (Xu et al., 2014). However, this was
not observed when MgCl2 was used as a cofactor (Figure 2).
This may be explained by the hydration capability of MgSO4
(heptahydrate) compared to MgCl2 (hexahydrated), acting as
an inhibitor of calcite by occlusion of Mg inside the platelet,
leading a less hydratated form of CaCO3 (Nielsen et al., 2016).
The transition of ACC to calcite and aragonite in Mollusk
is modulated by Mg2+ ions and SMPs (Ma and Feng, 2015).
In our study, Ps19 was able to induce well-formed aragonite
platelets, being the bigger aragonite crystal those found at
100 mM MgCl2 (Figure 2). However, the inhibition observed
at 40 mM MgCl2, and the less structured aragonite crystal at
100 mM MgCl2 when 1.2 µg·µL-1 Ps19 was used (Figure 2D, H),
suggest that Ps19 can act as an inhibitor of crystal growth
besides its role as a crystallization inductor, this dual role may
be ion and protein concentration-dependent as has been
suggested previously (Arroyo-Loranca et al., 2020). Similar
results have been observed in Prismalin-14, Pif97, KRMP-3,
and rPNU9 from P. fucata (Suzuki et al., 2004; Bahn et al., 2015;
Liang et al., 2016; Kong et al., 2019).
SMPs related to prismatic layer growth of the shell
have been found to form strictly calcite crystal at low CaCl2
concentration and to be able to inhibit aragonite platelet
growth, e.g. Prisilkin-39 from P. fucata (Kong et al., 2009).
However, Ps19 have been proved previously to produce cal-
cite (Arroyo-Loranca et al., 2020) at low CaCl2. Interestingly,
putative inhibition zones of crystal growth were observed in
this study at high CaCl2 concentration and 0.7 µg·µL-1 of Ps19
(Figure 3C), a behavior also been observed in other SMPs
such as rPif97 from P. fucata (Bahn et al., 2015), suggesting
that calcite crystal deposition may be protein dose-depen-
dent. The capability of Ps19 to produce calcite and aragonite
using dierent ions suggest that Ps19 is associated to the
formation of the prismatic and nacre layer in P. sterna, as has
been described in other SMPs such as Lys-rich matrix protein
family from P. fucata (Liang et al., 2016) and nacrein from the
Pacic oyster Crassostrea gigas (Song et al., 2014).
Besides modulation by a single ion, the growth of calcite
and aragonite is dependent on the Mg2+ and Ca2+ molar ratio
(Loste et al., 2003). An increase in the Mg2+ molar ratio leads
to the growth of a hydrated phase of CaCO3 at the surface
of the solution, while at the base of the crystallization assay
produces calcite and aragonite from 2:1, 3:1 and 4:1 of Mg:Ca
(Loste et al., 2003). For Ps19, a molar ratio of 1:1 leads to the
production of a thin lm and scarce crystal growth with
undened structures at both concentration, 40 and 100 mM,
however calcitic and aragonite structures were visible at 0.7
and 1.2 µg·µL-1 of Ps19, these results lead to the hypothesis
that Ps19 may react with the salts and inhibit the crystal
growth of either calcite or aragonite, and the crystals formed
are the result of dehydrated spots in the crystallization assay,
however further experiments are required corroborate this
hypothesis.
CONCLUSIONS
In contrast to other SMPs described in Mollusk, which promo-
te one kind of calcium carbonate polymorph (e.g. aragonite
or calcite), Ps19 is a novel protein capable to induce calcite
and aragonite crystals in a dose-dependent manner at die-
rent ion concentrations, acting as a promoter of aragonite in
presence of MgCl2 and as a promoter of calcite in presence of
CaCl2, suggesting that Ps19 may play an important role in the
prismatic and nacre layer of the shell of P. sterna.
Figure 8. Raman spectra of ACC polymorphs by using Ps19 protein
and MgSO4 as a cofactor. Calcium carbonate crystals growth in vitro in the
presence of A) 100 mM MgSO4 Na2CO3 or B) 40 mM MgSO4 Na2CO3. Color
line indicate salts preparations, red line: salt without protein added, black:
salt in the presente of 0.2 µg·µL-1, blue: salt in the presence of 0.7 µg·µL-1,
brown: salt in the presence of 1.2 µg·µL-1. Color arrows indicate the peaks
for each salt preparation.
Figura 8. Espectros Raman de polimorfos de ACC usando proteína
Ps19 y MgSO4 como cofactor. Crecimiento de cristales de carbonato de
calcio in vitro en presencia de A) 100 mM de MgSO4 Na2CO3, B) 40 mM de
MgSO4 Na2CO3 La línea de color indica preparaciones de sales, línea roja:
sal sin proteína añadida, negra: sal en presencia de 0.2 µg·µL-1, azul: sal en
presencia de 0.7 µg·µL-1, marrón: sal en presencia de 1.2 µg·µL-1. Las echas
de color indican los picos para cada preparación de sal.
144 Volume XXV, Issue 2
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144
ACKNOWLEDGMENTS
The authors are grateful to Delia Irene Rojas Posadas for her
technical assistance, as well as to M.C. Beatríz Adriana Rivera
Escoto for the measurements with the Raman spectrometer
from the National Laboratory for Nanoscience and Nanote-
chnology Research (LINAN) at IPICYT, A.C.; also to CONACyT
for the fellowship grant number 358437.
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