Role of calcium phosphate nanoclusters in the control of
calcification
Carl Holt
1
, Esben S. Sørensen
2
and Roger A. Clegg
1
1 Hannah Research Institute, Ayr, UK
2 Protein Chemistry Laboratory, Department of Molecular Biology, University of A
˚
rhus, Denmark
Many biological fluids, including blood, milk, extracel-
lular fluid, saliva, urine, synovial fluid and cerebrospi-
nal fluid, are usually supersaturated with respect to
hydroxyapatite (HA) [1–5], but generally remain stable.
Nevertheless, dystrophic calcification does occur, and
vascular calcification or stone-forming biofluids, for
example, have serious consequences for human health.
Genetic ablation and other experiments on individual
serum proteins have demonstrated the importance of
serum fetuin A (FETUA), osteopontin (OPN) and
matrix Gla protein (MGP) for inhibiting the precipita-
tion of calcium phosphate (CaP) in serum and prevent-
ing ectopic calcification of soft tissues [6–8]. A
metastable, colloidal, complex of CaP with FETUA,
MGP and secretory phosphoprotein 24 (SPP-24) forms
when the serum is destabilized [9,10], but the physio-
logical mechanism is still unclear.
Milk provides an example of a biofluid that seldom
forms CaP precipitates or causes dystrophic calcifica-

of nanocluster formation identified the factors of importance in determin-
ing the equilibrium size of the core, and showed how a nanocluster solution
could be thermodynamically stable yet supersaturated with respect to the
mineral phase of bones and teeth. It is suggested that the ability of some
secreted phosphoproteins to form nanoclusters is physiologically important
for the control or inhibition of calcification in soft and mineralized tissues,
the extracellular matrix and a wide range of biofluids, including milk and
blood.
Abbreviations
ACP, amorphous calcium phosphate; CaP, calcium phosphate; CPN, calcium phosphate nanocluster; DCPD, di-calcium phosphate di-hydrate;
DMP1, dentin matrix acidic phosphoprotein 1; FETUA, fetuin A; HA, hydroxyapatite; MGP, matrix Gla protein; OCP, octacalcium phosphate;
OPN, osteopontin; PC, phosphate centre; pS, phosphoseryl residue; RBP, riboflavin-binding protein; SAXS, small-angle X-ray scattering;
SCPP, secretory calcium-binding phosphoprotein; SP, secreted phosphoprotein; SPP-24, secretory phosphoprotein 24.
2308 FEBS Journal 276 (2009) 2308–2323 ª 2009 The Authors Journal compilation ª 2009 FEBS
a
S1
-, a
S2
- and b-caseins. Understanding the sequestra-
tion process has been furthered through studies with
short casein phosphopeptides containing a PC. Thus,
the 25-residue N-terminal b-casein tryptic phosphopep-
tide (b-casein 1–25) sequestered CaP to form a calcium
phosphate nanocluster (CPN) [12–14] with a core of
amorphous, acidic and hydrated calcium phosphate
(ACP) of radius 2.4 nm surrounded by a shell of about
50 phosphopeptides with a thickness of 1.6 nm. Ini-
tially, it was thought that the CPNs were metastable
particles in a state of arrested precipitation, but it was
later shown that they were equilibrium particles with a

tions is an involvement in the control of mineralization
processes [21,22].
Results
Thermodynamics of CPN formation
Doc. S1 (see Supporting information) provides addi-
tional details of the treatment. The chemical formula
of an electroneutral CPN can be written as a multiple
of an empirical formula, or ‘monomer’ containing a
single PC:
Ca
R
Ca
H
R
H
ðP
i
Þ
R
P
ðH
2
OÞ
R
W
ðPep À PCÞ
1
hi

j

OÞð2Þ
where 3y ⁄ (2 + y) is the mole fraction of P
i
in the
di-anionic form. The empirical chemical formula of
CaP can then be used to define a type of solubility
constant K
S
as an ion activity product. In a dilute
solution in which the activity of water is effectively
unity:
K
S
¼ a
1
Ca
2þ
a
y
HPO
2À
4
a
ð2À2yÞ=3
PO
3À
4
ð3Þ
K
S

À3A
core
DG
o
core

3V
core
4p

1=3
ð4Þ
where V
core
is the empirical formula volume of CaP,
k ¼ð36pV
2
core
Þ
1=3
, A
core
is the core surface area per PC,
DG
seq
is the free energy of sequestration of the core by
the shell of peptides, a
1
and a
s

ration is faster than the rate of sequestration, the
nanoclusters cannot form and a metastable solution
results. Certain partial SP sequences have been identi-
fied as the starting point of controlled crystal growth
in the extracellular matrix of mineralized tissues. These
include long phosphorylated sequences in, for example,
phosphophoryn, the C-terminal sequence of OPN and
the N-terminal sequence of dentin matrix acidic phos-
phoprotein 1 (DMP1) [27,28] and long sequences of
Glu residues in, for example, integrin-binding sialo-
phosphoprotein II [29]. When a sequence that can
sequester ACP and a sequence that can accelerate the
maturation of ACP into HA are both present in a
given SP, the competing reactions of ACP maturation
and ACP sequestration may make the formation of
CPNs as the equilibrium product more difficult or even
impossible. The formation of the nanocluster solution
requires not only that maturation of the ACP should
be prevented, but also a stoichiometric excess of the
phosphopeptide over CaP. If [p] molÆL
)1
of P
i
can
precipitate as ACP from the initially supersaturated
solution, the condition for thermodynamic stability is
a ¼
½p
f ½PPR
P

those found in phosphophoryn and the C-terminal half
of OPN and N-terminal part of DMP1, have been
shown to promote the maturation of ACP into
more crystalline phases, and so were discounted as
CPN-forming sequences. A minor PC pattern involves
three or more repeats of a primary kinase recognition
triplet SXE (MGP) or SD[E,pS] (OPN). When the
aligned orthologue sequences were examined (Doc. S2),
it was found that not all PCs were conserved, particu-
larly when a protein contained more than one PC. For
example, the N-terminal half of bovine OPN contained
all three PCs coded by exons 3, 5 and 6. The last two
were not as highly conserved as the first, but none of the
orthologues had fewer than two PCs.
Table 1. Identified PC sequences formed by the action of the Golgi
kinase and casein kinase 2 on selected secreted phosphoproteins.
CSN1S1, a
S1
-casein; CSN1S2, a
S2
-casein; CSN2, b-casein; IBSP-II,
integrin-binding sialophosphoprotein II; MEPE, matrix extracellular
bone phosphoglycoprotein. Potential sites of phosphorylation are
shown in bold.
Protein Species Swiss-Prot No. PC
a
SCPPs
OPN Cow P31096 6- TSSGSSEEKQ -15
42- QNSVSSEETD -51
99– SDESHHSDES -108

oughly tested of the predictors of partial or complete
disorder in proteins. It continues to perform well in
comparative tests with more recent methods [31], and
is one of the components in the most recent meta pre-
dictor, metaPrDOS [32]. According to PONDRÒ pre-
dictions, the positions of PC sequences in the SPs in
Table 1 were, with the exception of the globular pro-
tein riboflavin-binding protein (RBP), disordered, and
had disordered flanking sequences (Fig. 1A,B). The PC
motif of RBP was disordered and is undefined in the
crystal structure [33], but its N-terminal flanking
sequence was correctly predicted to be ordered. The
prediction for FETUA indicated a folded N-terminal
sequence containing the two cystatin-like domains, but
a flexible C-terminal half in which the PC lies. The
result for SPP-24 was the least clear-cut with only
short disordered sequences flanking the PC. Essentially
the same results were obtained by the top-idp predic-
tor [34], with the notable exception that SPP-24 was
borderline stable near the PC and stable in its flanking
sequences (Fig. 1C), but the metaPrDOS predictor [32]
agreed better with the PONDRÒ result for this protein
(Fig. 1D). All methods were in agreement in showing
that OPN has little or no stable conformation, and
hence can be described as a worm-like, or rheomorphic
[35], chain.
With the exception of proline-rich protein 1, all
other members of the SCPP paralogous group identi-
fied by Kawasaki and Weiss [24,36] were predicted by
PONDRÒ to be flexible over a substantial fraction of

The thermogram shown in Fig. 3 shows an almost per-
fectly smooth increase in specific heat with temperature
in accord with the SAXS observations of a worm-like
chain and consistent with the low chemical shift
dispersion in
1
H-NMR spectra of OPN [25].
Binding of Ca ions to OPN 1–149
Three pK values and three Ca ion association con-
stants were allowed to vary during the fitting to the
experimental isotherms of the b-casein 1–25 peptide,
and the resulting fitted curves are shown in Fig. 4. The
three Ca ion association constants obtained were 3000,
400 and 30 m
)1
. The single phosphoseryl residue (pS)
had an effective pK value of 6.0 and the cluster of
three pS residues ionized with a pK value of 7.2. The
OPN 1–149 isotherm, also shown in Fig. 4, was fitted
by two Ca ion association constants of 3000 (dianionic
phosphate) and 30 m
)1
but, because it does not have
the triplet of pS residues, two pK values of 6.4 and 5.0
were required.
Formation of OPN 1–149 nanoclusters
OPNmix and OPN 1–149 were able to sequester CaP
to form nanoclusters, but OPN could not, suggesting
that the extended phosphorylated sequences in the
C-terminal half either were too large to form PCs or

redispersion was not achieved, even after 4 months.
These experiments demonstrated that, like the casein
CPNs, the OPN 1–149 CPNs can be formed by either
a forward reaction from a supersaturated solution or
by a back reaction from a two-phase system containing
a precipitate of ACP and sufficient sequestering pep-
tide to convert all the ACP to CPNs. Neither casein
nor OPN phosphopeptides could form the nanoclusters
from partially matured ACP.
Characterization of OPN nanoclusters
SAXS of OPN 1–149 nanoclusters prepared by the
urea ⁄ urease method
The results of the SAXS measurements on CPN subs-
amples, measured as a function of time after the addi-
tion of urease, are summarized in Fig. 5A,B. The first
AB
C
D
Fig. 1. Prediction of disorder as a function of residue position in SPs having known or potential PC sequences. The positions of known or
predicted PCs in the sequence are shown as full lines. (A) PONDRÒ predictions for SCPPs in Table 1. (B) PONDRÒ predictions for the other
secreted phosphoproteins in Table 1. (C) TOP-IDP predictions for h-OPN and h-SPP-24 plotted as the midpoint of a window of 51 residues.
(D) metaPrDOS predictions for h-OPN and h-SPP-24.
Calcium phosphate sequestration by osteopontin C. Holt et al.
2312 FEBS Journal 276 (2009) 2308–2323 ª 2009 The Authors Journal compilation ª 2009 FEBS
two subsamples were taken after 17 min, when the pH
was 6.82, and after 50 min, when the pH was 6.87,
but, by the third sample, the pH was essentially con-
stant and close to 7.0. Strongly scattering spherical
particles formed from an initial state dominated by the
scattering of a statistical polymer but, after about

to ACP at pH 5.5. All other results in Fig. 6A were
recorded after the final pH value of 7.0 was attained.
A progressive loss of colloidal particles at the expense
of the CPN component occurred as the solution
matured. The intensity distribution of a similar solu-
tion that had been stored at ambient temperature and
pH 7 for 1 day showed that the colloidal particles were
nearly absent. In another experiment, CPNs prepared
with a mixture of casein phosphopeptides [38] by the
simple mixing method were compared with those made
by the urea ⁄ urease method. The turbidity A
1 cm
600 nm
ÀÁ
of
Fig. 2. Kratky plots of the SAXS of OPN (in 20 mM P
i
buffer,
pH 7.0, ionic strength 80 m
M) and of OPN 1–149 (in the CaP dilu-
tion buffer used in the nanocluster experiments). Fitted curves are
from the worm-like chain model. Each set of results has been
scaled by the mean square radius of gyration determined by the
fitting procedure.
Fig. 3. Normalized differential scanning calorimetry thermogram of
OPN 1-149 at pH 7.0.
Fig. 4. Ca-binding isotherms of b-casein 1–25 as a function of pH
and of OPN 1–149 at pH 7.0.
C. Holt et al. Calcium phosphate sequestration by osteopontin
FEBS Journal 276 (2009) 2308–2323 ª 2009 The Authors Journal compilation ª 2009 FEBS 2313

)1
Æcm. According to
the Henry equation [39], it corresponds to a f potential
of )15.4 mV.
A
B
C
D
Fig. 5. Study by SAXS of the maturation of nanoclusters prepared with OPN 1–149 by the urea ⁄ urease method. (A) Effect of time on the
radius of gyration determined by the Guinier method. (B) Normalized, q
2
-weighted SAXS of the nanoclusters diluted to 5 mg Æ mL
)1
after the
given times. (C) Model of the scattering of the matured nanocluster solution as a mixture of scattering from copolymer micelle-like nanoclus-
ters and free peptide. The scattering of the nanoclusters was obtained by subtracting the scattering of the free peptide from the total scat-
tering. Model calculations used the parameters b = 0.07 nm, A
core
= 0.25 nm
2
, r
o
= 12.5 nm, b = 0.35. (D) Representation of an OPN 1–149
nanocluster. An eighth section of the spherical core of ACP is shown. Surrounding the core is a shell of OPN 1–149 molecules, each
anchored to the core through its three PCs. For clarity, only one phosphopeptide molecule is shown. The mesh illustrates the position of the
surface of shear, which determines the hydrodynamic radius of the nanocluster. The diagram is scaled to give approximately correct impres-
sions of the relative magnitudes of A
core
, r
g,peptide

were used, and peptide binding
was calculated on the assumption that all the peptides
in the OPNmix sample had the same binding isotherm
as OPN 1–149. The complete model of ionic equilibria
was then used to calculate the composition of an equi-
librium diffusate, so that it could be compared with
the composition of the experimental ultrafiltrate
A
B
Fig. 6. Intensity distribution curves derived from the dynamic light
scattering measurements. (A) Unfiltered nanocluster solution
prepared with b-casein (f1–25) by the urea ⁄ urease method from an
initial pH of 5.5 to a final pH of 7.0. The larger particles observed at
pH 5.5 are probably colloidal ACP formed during mixing, which
gradually dissolve at the expense of the nanoclusters formed above
pH 6. (B) Mature OPN 1 149 nanoclusters.
A
B
C
Fig. 7. Calculated properties of OPNmix nanocluster solutions. (A)
Comparison of calculated ultrafiltrate concentrations of P
i
, Ca and
free Ca
2+
with experimental values shown as symbols. (B) Calcu-
lated fraction of reacted PCs. (C) Log of the saturation index versus
pH for DCPD, OCP and HA.
C. Holt et al. Calcium phosphate sequestration by osteopontin
FEBS Journal 276 (2009) 2308–2323 ª 2009 The Authors Journal compilation ª 2009 FEBS 2315

-casein 59–79 and b-cas-
eins 1–25 and 1–42. The results from the present work
utilized a peptide of 149 residues, and it is most likely
that the individual micellar CaP particles comprise the
core of equilibrium complexes formed from proteins of
more than 200 residues. It may be concluded that the
length of the peptide or protein is not an important
consideration. The OPN plasmin peptide has no signif-
icant sequence similarity to any casein sequence out-
side of the PCs. Flanking sequences of all the SPs in
Table 1 are deficient in hydrophobic residues and Cys,
and so they tend to have a low degree of sequence
complexity and favour an unfolded conformation.
On the larger scale, all PC-containing SCPPs and
the non-SCPPs proline-rich basic phosphoprotein 4
and MGP are known or predicted to be unfolded over
most or all of their length. The absence of a globular
structure close to the surface of the core allows a
higher density of PCs to bind to the surface, and so
clearly a fully globular protein is at a disadvantage.
The unfolded conformation may also allow a faster
rate of CaP sequestration, which may be of importance
when the rate of maturation of ACP nuclei is compa-
rable with the rate of sequestration. Nevertheless, it
can be envisaged that a globular domain, if it has an
extended, flexible, linker sequence connecting it to a
PC, could be just as effective as a natively unfolded
protein or short peptide. FETUA, with two cystatin-
like domains in the N-terminal half, and SPP-24, with
one, are predicted to have part of their sequence

face was about one-third of its value in free solution,
and this can be understood qualitatively if it is
assumed that the peptide is attached to the surface
through the three PCs (Fig. 5D). Compared with the
casein CPNs, the core CaP is more basic, correspond-
ing to the empirical chemical formula of TCP, and
nearly four times larger, but the molar ratios of Ca or
P
i
to PC were calculated to be the same. Most proba-
bly, the core is simply more hydrated. According to
Eqn (4), the size is determined mainly by the ratio of
the free energy of sequestration to the free energy of
formation of the bulk core phase and the core surface
area per PC. The latter was found to be 0.25 nm
2
,
which is about one-quarter of that for the
b-casein 1–25 CPN, and so this alone could account
Calcium phosphate sequestration by osteopontin C. Holt et al.
2316 FEBS Journal 276 (2009) 2308–2323 ª 2009 The Authors Journal compilation ª 2009 FEBS
for the difference. It is more probable that the differ-
ence in chemical composition and hydration in the
core affects the two free energy terms equally, so that
their ratio is unchanged.
Notwithstanding the difference in hydration in the
core, it is most probable that the core is amorphous,
similar to CaP in casein micelles and the core CaP of
casein CPNs, otherwise the particles would not have
equilibrated to a path-independent constant size.

pic deposit of ACP does form, it can be removed by
an excess of the sequestering protein or peptide.
Third, in contact with hard tissue, the nanocluster
solution cannot cause demineralization and could
indeed act as a reservoir of CaP for crystal growth
or tissue remineralization. Fourth, Eqn (5) places no
upper limit on the concentrations of Ca and P
i
in
the fluid. For example, the free Ca ion concentra-
tions and supersaturation with respect to HA in milk
A
BC
Fig. 8. Schematic drawing of the alternative fates of ACP nuclei formed from a supersaturated solution. (A) In the absence of a competent
sequestering peptide [i.e. a in Eqn (5) is infinite], ACP nuclei grow and mature into a crystalline or poorly crystalline calcium phosphate;
under physiological conditions, the final state is usually poorly crystalline OCP or HA or, in the case of tooth enamel, highly crystalline HA.
(B) In the presence of a stoichiometric excess or equivalence of PCs (0 < a £ 1), a thermodynamically stable solution of CPNs may form if
all the CaP is sequestered by the competent SPs. The CPNs have a defined composition and size at equilibrium. If some of the nuclei
escape sequestration to grow and mature to a poorly crystalline state, they cannot subsequently form the equilibrium nanoclusters. (C) In
the presence of a substoichiometric concentration of competent SPs (1 < a < ¥), the growth and maturation of the ACP nuclei may be slo-
wed to give a metastable colloidal suspension or precipitate of complexes of variable stoichiometry, size and degree of crystallinity.
C. Holt et al. Calcium phosphate sequestration by osteopontin
FEBS Journal 276 (2009) 2308–2323 ª 2009 The Authors Journal compilation ª 2009 FEBS 2317
remain comparable with those in blood, even though
the total Ca concentration in milk may be two
orders of magnitude higher. Fifth, there is scope for
an exquisite degree of control of mineralization
through the degree of phosphorylation of the compe-
tent SPs, particularly when, as in OPN and DMP1,
they have opposing functional subsequences that can

which a < 1, the product of interaction between
FETUA and CaP may be a thermodynamically
stable serum.
In conclusion, the present findings provide the first
description of equilibrium CPN formation by a phos-
phopeptide that is widely distributed among extant
species, tissues and biofluids, and was one of the earli-
est members of the paralogous group of SCPPs to
emerge in the late Cambrian together with CaP tissue
mineralization [23,24]. Other members of the paralo-
gous group and some non-SCPPs (Table 1) have been
identified as having the potential PCs and predicted
flexible conformations that are the hallmark of CPN-
forming phosphopeptides. One or more of these pro-
teins is physiologically important in the tissues of
bone, dentine, cementum and osteoid, or is secreted
into biofluids, such as blood, milk, saliva and urine.
Our contention is that among the physiologically
important functions of the non-casein SPs of Table 1
are presently unrecognised ones that involve the
formation of thermodynamically stable complexes such
as CPNs.
Experimental procedures
Sequence analyses
Identification of PCs in secreted phosphoproteins
Searches for PCs were made by manual and automated
methods in the sequences of SPs, known to the authors to
be involved in CaP mineralization processes, using the Uni-
Prot (=Swiss-Prot + TrEMBL) database on the ExPASy
server () of the Swiss Institute of

shorter regions. Predictions were checked against the more
recent predictor from the same group, TOP-IDP [34], and
human OPN and SPP24 sequences were submitted to a
recent meta predictor, metaPrDOS, which, on this occa-
sion, employed an optimized combination of five predic-
tors [32].
Fractionation of the OPNmix sample by gel filtration
chromatography
An OPN fraction (OPNmix) was isolated from bovine
milk by the method of Sorensen et al. [26] It comprised
better than 95% OPN or OPN peptides, phosphorylated
to some degree at all sites. It was fractionated further by
Superdex 75 gel filtration chromatography using a Pharmacia
Calcium phosphate sequestration by osteopontin C. Holt et al.
2318 FEBS Journal 276 (2009) 2308–2323 ª 2009 The Authors Journal compilation ª 2009 FEBS
XK 16 column (Pharmacia Ltd., Sandwich, UK) with
a bed length of 64 cm at a flow rate of 0.3 mLÆmin
)1
.
The sample of 10 mg was dissolved in 1 mL of elution
buffer (50 mm phosphate, 300 mm NaCl, 0.02% NaN
3
,
pH 7.0) and dialysed overnight against 120 mL of elution
buffer before loading on to the column. Fractions of
1 mL were collected and examined by SDS–Mops gel elec-
trophoresis before pooling into four fractions (F1, F2, F3a
and F3b) which were dialysed exhaustively against deion-
ized water, freeze dried and the recovered masses recorded.
The procedure was repeated as required to collect enough

and the phospho-
peptide, the concentration gradients generated and their
persistence during inefficient mixing can allow the inital
ACP to mature into a more stable state which can no
longer form equilibrium CPNs. Even when this does not
happen, there can be an overshoot past the equilibrium
state to generate colloidal metastable complexes of ACP
and the phosphopeptide, and the subsequent re-equilibra-
tion can take days or weeks to complete. To circumvent
these problems, we developed the urea ⁄ urease method [12].
In this method, the reagents are mixed together to give an
initial undersaturated solution with a low pH, typically
5–5.5. The pH is then increased homogeneously by hydroly-
sis of a precise number of moles of urea using urease to
catalyse the reaction, producing the strong base ammonia
and weak carbonic acid. The amount of urea determines
the final pH (typically 6–8) and the amount of enzyme can
vary the time taken to approach the target pH from 2 min
to 2 h. Rapid attainment of the target pH is followed by
several hours during which the nanoclusters grow to their
equilibrium size [13].
Nanoclusters were prepared by the urea ⁄ urease method
[12] using either the OPNmix or OPN 1–149 (fraction F3a)
and magnesium-free Buffer A [13]. The standard concentra-
tion of peptide was either 25 or 30 mgÆmL
)1
, which corre-
sponds to a similar molar concentration to that used in
previous work with caseins. It did not prove possible to
find suitable conditions for CPN formation with OPN or

½Ca
c
¼ f ½PP R
Ca
a þð1 À aÞ

t
Ca
ð½Ca
2þ
; pHÞ
ÀÁ
ð6Þ
where [PP] is the phosphopeptide concentration and the
function

t
Ca
ð½Ca
2þ
; pHÞ is the phosphopeptide binding iso-
therm. From these values, the diffusible concentrations
were calculated, and hence the composition of the ultrafil-
trate was obtained from the Donnan equilibrium across the
membrane [46].
Equation (6) requires the calculation of Ca ion binding
to the free peptide at any pH in the range 5.0–8.0. A
semi-empirical model was used to describe the binding
isotherms obtained previously for the b-casein 1–25 phos-
phopeptide in this pH range, and the same model was

pH values between 5.0 and 7.5. They were allowed to equil-
ibrate before ultrafiltration through a 0.5 mL centrifugal
concentrator with a molecular mass cut-off of 10 000 Da,
(Vivascience AG, Hanover, Germany) using a centripetal
field of 5000 g for 15 min. The concentrations of Ca, free
Ca
2+
and P
i
in the ultrafiltrate and starting solution were
determined as described previously [13]. The peptide-free
ultrafiltrate composition was then used to calculate the
ionic equilibria [5] and ion activity product for CaP,
according to Eqn (3), for y in the range 0–1.
Small-angle X-ray scattering
Measurements were made on station 2.1 at the CCLRC
Daresbury Laboratory (Warrington, Cheshire, UK), as
described previously [13]. The radii of gyration and the
intercept at q = 0 were determined by a Guinier plot of
ln(I) versus q
2
. Scattering curves were normalized by divid-
ing by the Guinier intercept and weighted by q
2
to empha-
size the low-intensity features (Kratky plot). The scattering
of OPN and OPN 1–149 in free solution was fitted to a
worm-like chain model [47,48]. Chain stiffness was mea-
sured by means of the Kuhn segment length b, which is
the peptide bond length in an equivalent freely jointed

tion function by means of the Multiple Narrow Modes
algorithm in the instrument’s software gave an intensity-
weighted size distribution. The mean hydrodynamic radius
(

r
h
) was calculated from the diffusion coefficient using the
Stokes–Einstein equation. Electrophoretic mobilities were
measured by the phase analysis method in the disposable
single-use cells supplied with the instrument. The zeta
potential (f) was calculated from the electrophoretic mobil-
ity in a unit field (u
e
) using the Henry equation [39], as the
particle size is between the Hu
¨
ckel and Smoluchowski limit-
ing formulae.
An OPN 1–149 CPN sample was prepared at pH 7 by
the rapid urea ⁄ urease method at a peptide concentration of
25 mgÆmL
)1
and matured for 1 day. It was diluted with
dilution buffer to a concentration of 5 mgÆmL
)1
, filtered
and the electrophoretic mobility and diffusion coefficient
were determined. The urea ⁄ urease method was also used to
make CPNs with b-casein (f1-25), and a study was made of

Calcium phosphate sequestration by osteopontin C. Holt et al.
2320 FEBS Journal 276 (2009) 2308–2323 ª 2009 The Authors Journal compilation ª 2009 FEBS
Ste 160, Indianapolis, IN 46268, USA; Tel: 00 1 317
280 8737; E-mail: ). VL-
XT is copyright ª1999 by the WSU Research Founda-
tion (Pullman, WA, USA), all rights reserved.
PONDRÒ is copyright ª2004 by Molecular Kinetics,
all rights reserved.
References
1 Robertson WG & Marshall RW (1979) Calcium mea-
surements in serum and plasma – total and ionized.
CRC Crit Rev Clin Lab Sci 11, 271–304.
2 Glinkina IV, Durov VA & Mel’nitchenko GA (2004)
Modelling of electrolyte mixtures with application to
chemical equilibria in mixtures – prototypes of blood’s
plasma and calcification of soft tissues. J Mol Liquids
110, 63–67.
3 Silwood CJL, Grootveld M & Lynch E (2002) H-1
NMR investigations of the molecular nature of low-
molecular-mass calcium ions in biofluids. J Biol Inorg
Chem 7, 46–57.
4 Gyory AZ & Ashby R (1999) Calcium salt urolithiasis –
review of theory for diagnosis and management. Clin
Nephrol 51, 197–208.
5 Holt C, Dalgleish DG & Jenness R (1981) Inorganic
constituents of milk. 2. Calculation of the ion equilibria
in milk diffusate and comparison with experiment. Anal
Biochem 113, 154–163.
6 Price PA & Lim JE (2003) The inhibition of calcium
phosphate precipitation by fetuin is accompanied by the

1035–1039.
13 Little EM & Holt C (2004) An equilibrium thermody-
namic model of the sequestration of calcium phosphate
by casein phosphopeptides. Eur Biophys J Biophys Lett
33, 435–447.
14 Holt C, Timmins PA, Errington N & Leaver J (1998) A
core-shell model of calcium phosphate nanoclusters sta-
bilized by beta-casein phosphopeptides, derived from
sedimentation equilibrium and small-angle X-ray and
neutron-scattering measurements. Eur J Biochem 252,
73–78.
15 Holt C, Vankemenade M, Nelson LS, Sawyer L, Har-
ries JE, Bailey RT & Hukins DWL (1989) Composition
and structure of micellar calcium-phosphate. J Dairy
Res 56, 411–416.
16 Holt C (2004) An equilibrium thermodynamic model of
the sequestration of calcium phosphate by casein
micelles and its application to the calculation of the
partition of salts in milk. Eur Biophys J Biophys Lett
33, 421–434.
17 Holt C, de Kruif CG, Tuinier R & Timmins PA (2003)
Substructure of bovine casein micelles by small-angle
X-ray and neutron scattering. Colloids Surf A
Physicochem Eng Asp 213, 275–284.
18 Holt C, Hasnain SS & Hukins DWL (1982) Structure
of bovine-milk calcium-phosphate determined by x-ray
absorption-spectroscopy. Biochim Biophys Acta 719 ,
299–303.
19 Kruif CGd & Holt C (2003) Casein micelle structure,
functions and interactions. In Advanced Dairy

sites. Protein Sci 4, 2040–2049.
27 Hunter GK, Hauschka PV, Poole AR, Rosenberg LC
& Goldberg HA (1996) Nucleation and inhibition of
hydroxyapatite formation by mineralized tissue pro-
teins. Biochem J 317, 59–64.
28 He G, Ramachandran A, Dahl T, George S, Schultz D,
Cookson D, Veis A & George A (2005) Phosphoryla-
tion of phosphophoryn is crucial for its function as a
mediator of biomineralization. J Biol Chem 280, 33109–
33114.
29 Hunter GK & Goldberg HA (1994) Modulation of
crystal-formation by bone phosphoproteins – role of
glutamic acid-rich sequences in the nucleation of
hydroxyapatite by bone sialoprotein. Biochem J 302,
175–179.
30 Nancollas GH (1982) Phase transformation during pre-
cipitation of calcium salts. In Biological Mineralization
and Demineralization (Nancollas GH, ed.), pp. 79–99.
Springer-Verlag, Berlin.
31 Ferron F, Longhi S, Canard B & Karlin D (2006) A
practical overview of protein disorder prediction meth-
ods. Proteins Struct Funct Bioinform 65, 1–14.
32 Ishida T & Kinoshita K (2008) Prediction of disordered
regions in proteins based on the meta approach. Bioin-
formatics 24, 1344–1348.
33 Monaco HL (1997) Crystal structure of chicken ribofla-
vin-binding protein. EMBO J 16, 1475–1483.
34 Campen A, Williams RM, Brown CJ, Meng JW, Uver-
sky VN & Dunker AK (2008) TOP-IDP-scale: a new
amino acid scale measuring propensity for intrinsic dis-

uin, and matrix Gla protein: biochemical evidence for
the cancellous bone-remodeling compartment. J Bone
Miner Res 17, 1171–1179.
43 Price PA, Nguyen TMT & Williamson MK (2003) Bio-
chemical characterization of the serum fetuin–mineral
complex. J Biol Chem 278, 22153–22160.
44 Larkin MA, Blackshields G, Brown NP, Chenna R,
McGettigan PA, McWilliam H, Valentin F, Wallace
IM, Wilm A, Lopez R et al. (2007) Clustal W and clus-
tal X version 2.0. Bioinformatics 23, 2947–2948.
45 Jonassen I (1997) Efficient discovery of conserved pat-
terns using a pattern graph. Comput Appl Biosci 13,
509–522.
46 Holt C (1997) The milk salts and their interaction with
casein. In Advanced Dairy Chemistry (Fox PF, ed.), pp.
233–256. Chapman & Hall, London.
47 Kholodenko AL (1993) Scattering function for semiflex-
ible polymers – Dirac versus Kratky–Porod. Phys Lett
A 178, 180–185.
48 Potschke D, Hickl P, Ballauff M, Astrand PO &
Pedersen JS (2000) Analysis of the conformation of
worm-like chains by small-angle scattering: Monte-
Carlo simulations in comparison to analytical theory.
Macromol Theory Simul 9, 345–353.
49 Svaneborg C & Pedersen JS (2002) Form factors of
block copolymer micelles with excluded-volume interac-
tions of the corona chains determined by Monte Carlo
simulations. Macromolecules 35, 1028–1037.
50 Qi XL, Holt C, McNulty D, Clarke DT, Brownlow S &
Jones GR (1997) Effect of temperature on the second-