Vol.
167,
March
No.
16,
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
1990
761-766
HIGH-RESOLUTION SOLID-STATE NUCLEAR MAGNETIC RESONANCE SPECTRA OF DENTIN COLLAGEN
Dept.
Received
of
February
R. Fujisawa
and Y. Kuboki
Biochemistry, Hokkaido Sapporo,
School of University, 060 Japan
Dentistry,
7, 1990
Summary: Insoluble collagen of /ZJovine dentin was characterized by high-resolution solid-state 3C nuclear magnetic resonance (NMR) spectroscopy using a cross-polarization magic angle spinning procedure. A downfield shift was observed in the signal of hydroxyproline Cp compared with that in skin collagen, in the hydroxyproline structure. A ;y;;;;tgrg 3p distortion P NMR was detected in dentin collagen that was compatible with the presence of matrix-associated phosphoprotein.
Mineralized have
tissue
unique
collagens
properties (for
collagens
are
resistant
and
properties
are
collagens.
and
components
its
packed
readily to
Most
the
swell
in
chemistry
cross-links
covalent
with
(2).
These
and structure
of
revealed
various
mineralized
association
tissue denaturing
acids
have
of
tissue
mineralized even
approaches
approaches
of
mineralized revealed
more tightly
and colleagues using
not
collagen,
tissue
with
the
collagen
non-collagenous
(6-10).
Physical
diffraction
1).
dentin
unmineralized
to solubilization
related
of
with
see Ref.
Chemical
characteristics
mobility
do
especially
compared
a review
solvents,
(3-51,
collagens,
solid-state
have
have
revealed
tissue that
than
collagens.
collagen in
in
fully
unmineralized
done substantial nuclear
the
magnetic
molecular A study
with
mineralized tissues
work
packing
neutron bone was Torchia
(11).
on molecular
resonance
mobility
(NMR)
(12-15).
0006-291x/90 761
and
$1.50
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
167,
Their
No.
results
restrict
indicate
The
improved
the
was
introduced
its
early
the
and
In
NMR spectra signals chemical tissue
present of
were shifts
of
assigned were
mineralization backbone
of
the
spinning solid-state
structure
(16).
the
chemical (19,
we present
compared
to with
However,
in
was not Assignment
et
values
al.
(18),
with
the
20).
high-resolution
and bone insoluble according
shift
method and
(17).
by Saito
of
NMR,
resolution
each signal
polypeptides
paper,
dentin
of
collagen,
COMMUNICATIONS
molecular
was accomplished
some of
structure
the
the
protein
assignment
signals
and
sensitivity
to dentin
allow
related
secondary
of
RESEARCH
magic-angle
and
to study
collagen
they
of
cross-polarization
resolution
to
BlOPHYSlCAL
cross-linking
movement
application
good enough
AND
that
azimuthal
collagen.
of
BIOCHEMICAL
2, 1990
solid-state
collagens. Saito
et
those
of
al.
Some of
the
(181,
and
unmineralized
collagens.
MATERIALS AND METHODS Incisors and tibiae were Preparation of The Insoluble Collagens: were cleaned mechanically and collected from 2y-old calves, crushed in a liquid nitrogen mill (9). The crushed powders, having a diameter less than 0.3 mm, were washed with 4M guanidine/HCl, 50 mM Tris/HCl, pH 7.4, rinsed with water and The lyophilized powders were used as samples for lyophilized. NMR study. Aliquots of the dentin or bone powders were demineralized with 0.5 M EDTA, 50 mM Tris/HCl, pH 7.4 , containing protease inhibitors (21). The completeness of the demineralization was checked by measuring calcium content in the demineralized matrix. Demineralized powder was rinsed with water and lyophilized. Acid insoluble collagens were prepared from Achilles' tendon and skin of calves by a conventional procedure (22). These collagens were crushed into powder in a liquid nitrogen mill, rinsed with water and lyophilized. Solid-State NMR Spectra: 13C cross-polarization magic-angle spinning (CP-MAS) NMR spectra were recorded at 100.63 MHz with a uker MSL-400 spectrometer equipped with a CP-MAS accessory. 3 P CP-MAS NMR spectra were recorded at 161.98 MHz with the same equipment. Sample capsules were spun at 4KHz. Contact time was 1 ms, repetition time was 5s, and spectral width was 31.2 KHz. Spectra re accumulated 500 times to improve the signal-to-noise 733C, chemical ratio. shifts were referred to tetramethylsilane, and the P chemical shifts were referred to inorganic orthophosphate. 762
Vol.
167,
No.
RESULTS The dentin
2, 1990
BIOCHEMICAL
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
AND DISCUSSION high-resolution
solid-state
and bone collagen
reported
by
Saito
et
assigned
to
amino
acids
the
assigned
signal
are
Ala
Cp
is
known
polypeptide P-sheet
AND
(20, and
to 21).
collagen
were al.
essentially
listed with
spectra
obtained
similar
to
Some of
(18).
(Figs.
vary
NMR
1, 2).
The chemical helix
I.
the
spectra
13C signals
Chemical
in Table the
the
from
shift
values
of
shift
of
of
the
The chemical
secondary shift
structure
values
are14.9,lg.g
for and
were
a-helix, 17.6
ppm,
C
02--+180 160f80 PPm60 Fiqure 1. 13C CP-MAS NMR spectra of bone (A), decalcified (B) and skin (C) collagens. The spectra were recorded Bruker MSL-400 spectrometer at 100.63 MHz. Some of the were assigned to amino acids according to Saito et a1.(18). Figure dentin
2. (B)
13C CP-MAS NMR spectra and tendon (C) collagens.
763
of
dentin
(A),
LO 20 bone with a signals
decalcified
Vol.
167,
No.
BIOCHEMICAL
2, 1990
I. 13C chemical and unmineralized
Table
Tissues
Skin Tendon Demineralized bone Bone
Demineralized dentin Dentin
respectively.
The
collagen
were
collagen
helix.
Minor
in
good
significant
chemical
shifts
of
chemical
shift that
of in
than
in
the
shift peptide Pro
and
represent form.
29.3 30.5
25.5 25.3
17.6
70.1 70.7
58.9 58.9
42.7 42.9
37.6 38.6
29.5 29.1
24.9 25.2
17.5 17.6
70.7 70.5
59.2 59.3
42.8 42.5
38.8 39.6
29.7 30.2
25.2 25.1
17.6 17.5
Cp
chemical
is
the
was
the
possibility
is
that tight
Hyp
around change
the
Hyp the
can
the
Hyp residue.
(23).
residue
from
of
distortion
hydroxyproline packing 764
of
collagen.
The
Cp
a mineral
collagen
chemical of
By analogy
Hyp
bulky
Hyp
some
transition
of
with
after
be attributed
cis-trans
shift
residue
The
to
bone
tendon
signal
such
between
Even
in
Cp
with
typical
retained
in
residue
causes
molecular
still
that
bone
ppm downfield
I).
prominent
to
the
observed
a few
was
and
tissues.
(Table
less
a proline
possible
of
be
deviated
shift
to
in
the
value
tissue
known
of
the
dentin
and unmineralized
downfield
the
of
could
dentin
of
a distortion One of
shifts
with
comparable
including
by
37.2 38.7
downfield
was
interaction
distorted
42.8 43.1
change
Cp
,
58.9 59.5
shift
bonds Cp
70.8 71.1
shift
conformational Pro
Ala cp
unmineralized
dentin,
of
Pro cy
mineralized
The downfield to
Pro cp
agreement
downfield
COMMUNICATIONS
of mineralized collagens
differences
the
The
extent.
shifts tissue
RESEARCH
Pro GUY HYP ca ca cp HYP Co!,6
Hyp CB in
demineralization
BIOPHYSICAL
HYP cy
Ala
but
from
AND
is the
with
assumed typical is
(24). side
mineralized
to trans
a direct Another chain
was tissue
Vol.
167,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
I
60
Fiqure
3.
31P CP-MAS
spectrum was recorded 161.98 MHz.
collagen than
that
(11). of Pro
Certain also
Side
0 -20 PPm
-40
-60
NMR spectrum of decalcified dentin. with a Bruker MSL-400 spectrometer
movement
of
Hyp is
The at
more restricted
(15).
characteristics
observed
of
in carbonyl from
and bone collagen
(Figs.
not
20
chain
ppm was separated
were
40
resolved
into
dentin
carbon. the
A smaller
main signal
1,
and bone collaqens
at
As signals
2).
each amino acid,
signal
further
170-171
ppm in dentin
173-174
of
at
were
carbonyl analysis
carbons was not
attempted. Dentin
insoluble
phosphoprotein,
3).
was
confirmed
demineralized
The line
contains
phosphophoryn.
phosphoprotein completely
collagen
width
of
The by
dentin the
the
detecting
with
signal
covalently
attached
presence the
phosphate
31P solid-state
was large,
since
of
the in
NMR (Fig. the
phosphate
was bound to a macromolecule. ACKNOWLEDGMENTS The authors are grateful to the NMR laboratory, Faculty of Engineering, Hokkaido University, for NMR analysis, and to Dr. H. Saito, National Cancer Center Research Institute, for valuable scientific discussions. This study was supported by the Japanese 765
Vol.
167,
No.
BIOCHEMICAL
2, 1990
Ministry of for Scientific
Education, Research.
AND
Science
and
BIOPHYSICAL
RESEARCH
Culture
under
COMMUNICATIONS
a Grant-in-Aid
REFERENCES 1. 2.
A. (1984) In Extracellular Matrix Biochemistry (PieZ, Veis, X.A. and Reddi, A-H., Eds.), pp329-374, Elsevier, Amsterdam. Veis, A.and Schlueter, R.J. (1964) Biochemistry 3, 16501656.
3. 4. 5. 6.
Kuboki, Y., Takagi, T., Sasaki, S., Saito, S. and Mechanic, G.L. (1981) J. Dent. Res. 61, 159-163. Kuboki, Y., Tsuzaki, M., Sasaki, S., Liu, C.F. and Mechanic, G.L. (1981) Biochem.Biophys.Res.Commun.102, 119-126. Kuboki, Y., Takagi, T., Shimokawa, H., Oguchi, H., Sasaki, S. and Mechanic, G.L. (1981) Calcif. Tissue Res. 9, 107-114. Curley-Joseph, J. and Veis, A. (1979) J. Dent. Res. 58, 1625-1633.
7. 8.
Kuboki, Y., Fujisawa, R., Aoyama, K. and Sasaki, J. Dent. Res. 58, 1926-1932. Lee, S.L. and Veis, A. (1980) Calcif. Tissue Int.
S. (1979) 31,
123-
124. 9.
Fujisawa, (1984)
10.
Fujisawa,
R. and Takagi, T., Kuboki, Y. and Calcif. Tissue Int. 36, 239-242. R. and Kuboki, Y. (1988) Connect.
Sasaki,
S.
Tissue
Res.
L.C.,
J.
17,
231-238. 11.
Bonar, 181,
12.
Torchia, 104,
Lees,
S. and
Mook,
H.A.
(1985)
Mol.
Biol.
265-270.
D.A.
and
Vander
Hart,
D.L.
(1976)
J.
Mol.
Biol.
315-321.
Sarkar, S.K., Sullivan, C.E. and Torchia, D.A. (1983) J. Biol. Chem. 258, 9762-9767. 14. Sarkar, S.K., Sullivan, C.E. and Torchia, D.A. (1985) Biochemistry 24, 2348-2354. 15. Sarkar, S.K., Hiyama, Y., Niu, C.H., Young, P-E., Gerig, J.T. and Torchia, D.A. (1987) Biochemistry 26, 6793-6800. 16. Saito, H. (1986) Magn. Reson. Chem. 24, 835-852. 17. Schaefer, J., Stejskal, E.O., Brewer, C.F., Keiser, H.D. and Sternlicht, H. (1978) Archs. Biochem. Biophys. 190, 657-661. 18. Saito, H., Tabeta, R., Shoji, A., Ozaki, T., Ando, I. and Miyata, T. (1984) Biopolymers 23, 2279-2297. 19. Ando, I., Saito, H., Tabeta, R., Shoji, A. and Ozaki, T. (1984) Macromolecules 17, 475-461. 20. Saito, H., Tabeta, R., Asakura, T., Iwanaga, Y., Shoji, A., Ozaki, T. and Ando, I. (1984) Macromolecules 17, 1405-1412. 21. Butler, W.T. (1987) Methods in Enzymol. 145, 290-303. 22. Miller, E.J. and Rhodes, R.K. (1982) Methods in Enzymol. 82, 13.
33-41. 23.
24.
Keim, P., Vigna, R.A., Nigen, A.M., Morrow, J.S. and Gurd, F.R.N. (1974) J. Biol. Chem. 249, 4149-4156. Hata, M., Marumo, F. and Aoki, H. (1983) Report of the Research Laboratory of Engineering Material. Tokyo Institute of Technology 8, I-15.
766
167,
March
No.
16,
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
1990
761-766
HIGH-RESOLUTION SOLID-STATE NUCLEAR MAGNETIC RESONANCE SPECTRA OF DENTIN COLLAGEN
Dept.
Received
of
February
R. Fujisawa
and Y. Kuboki
Biochemistry, Hokkaido Sapporo,
School of University, 060 Japan
Dentistry,
7, 1990
Summary: Insoluble collagen of /ZJovine dentin was characterized by high-resolution solid-state 3C nuclear magnetic resonance (NMR) spectroscopy using a cross-polarization magic angle spinning procedure. A downfield shift was observed in the signal of hydroxyproline Cp compared with that in skin collagen, in the hydroxyproline structure. A ;y;;;;tgrg 3p distortion P NMR was detected in dentin collagen that was compatible with the presence of matrix-associated phosphoprotein.
Mineralized have
tissue
unique
collagens
properties (for
collagens
are
resistant
and
properties
are
collagens.
and
components
its
packed
readily to
Most
the
swell
in
chemistry
cross-links
covalent
with
(2).
These
and structure
of
revealed
various
mineralized
association
tissue denaturing
acids
have
of
tissue
mineralized even
approaches
approaches
of
mineralized revealed
more tightly
and colleagues using
not
collagen,
tissue
with
the
collagen
non-collagenous
(6-10).
Physical
diffraction
1).
dentin
unmineralized
to solubilization
related
of
with
see Ref.
Chemical
characteristics
mobility
do
especially
compared
a review
solvents,
(3-51,
collagens,
solid-state
have
have
revealed
tissue that
than
collagens.
collagen in
in
fully
unmineralized
done substantial nuclear
the
magnetic
molecular A study
with
mineralized tissues
work
packing
neutron bone was Torchia
(11).
on molecular
resonance
mobility
(NMR)
(12-15).
0006-291x/90 761
and
$1.50
Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol.
167,
Their
No.
results
restrict
indicate
The
improved
the
was
introduced
its
early
the
and
In
NMR spectra signals chemical tissue
present of
were shifts
of
assigned were
mineralization backbone
of
the
spinning solid-state
structure
(16).
the
chemical (19,
we present
compared
to with
However,
in
was not Assignment
et
values
al.
(18),
with
the
20).
high-resolution
and bone insoluble according
shift
method and
(17).
by Saito
of
NMR,
resolution
each signal
polypeptides
paper,
dentin
of
collagen,
COMMUNICATIONS
molecular
was accomplished
some of
structure
the
the
protein
assignment
signals
and
sensitivity
to dentin
allow
related
secondary
of
RESEARCH
magic-angle
and
to study
collagen
they
of
cross-polarization
resolution
to
BlOPHYSlCAL
cross-linking
movement
application
good enough
AND
that
azimuthal
collagen.
of
BIOCHEMICAL
2, 1990
solid-state
collagens. Saito
et
those
of
al.
Some of
the
(181,
and
unmineralized
collagens.
MATERIALS AND METHODS Incisors and tibiae were Preparation of The Insoluble Collagens: were cleaned mechanically and collected from 2y-old calves, crushed in a liquid nitrogen mill (9). The crushed powders, having a diameter less than 0.3 mm, were washed with 4M guanidine/HCl, 50 mM Tris/HCl, pH 7.4, rinsed with water and The lyophilized powders were used as samples for lyophilized. NMR study. Aliquots of the dentin or bone powders were demineralized with 0.5 M EDTA, 50 mM Tris/HCl, pH 7.4 , containing protease inhibitors (21). The completeness of the demineralization was checked by measuring calcium content in the demineralized matrix. Demineralized powder was rinsed with water and lyophilized. Acid insoluble collagens were prepared from Achilles' tendon and skin of calves by a conventional procedure (22). These collagens were crushed into powder in a liquid nitrogen mill, rinsed with water and lyophilized. Solid-State NMR Spectra: 13C cross-polarization magic-angle spinning (CP-MAS) NMR spectra were recorded at 100.63 MHz with a uker MSL-400 spectrometer equipped with a CP-MAS accessory. 3 P CP-MAS NMR spectra were recorded at 161.98 MHz with the same equipment. Sample capsules were spun at 4KHz. Contact time was 1 ms, repetition time was 5s, and spectral width was 31.2 KHz. Spectra re accumulated 500 times to improve the signal-to-noise 733C, chemical ratio. shifts were referred to tetramethylsilane, and the P chemical shifts were referred to inorganic orthophosphate. 762
Vol.
167,
No.
RESULTS The dentin
2, 1990
BIOCHEMICAL
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
AND DISCUSSION high-resolution
solid-state
and bone collagen
reported
by
Saito
et
assigned
to
amino
acids
the
assigned
signal
are
Ala
Cp
is
known
polypeptide P-sheet
AND
(20, and
to 21).
collagen
were al.
essentially
listed with
spectra
obtained
similar
to
Some of
(18).
(Figs.
vary
NMR
1, 2).
The chemical helix
I.
the
spectra
13C signals
Chemical
in Table the
the
from
shift
values
of
shift
of
of
the
The chemical
secondary shift
structure
values
are14.9,lg.g
for and
were
a-helix, 17.6
ppm,
C
02--+180 160f80 PPm60 Fiqure 1. 13C CP-MAS NMR spectra of bone (A), decalcified (B) and skin (C) collagens. The spectra were recorded Bruker MSL-400 spectrometer at 100.63 MHz. Some of the were assigned to amino acids according to Saito et a1.(18). Figure dentin
2. (B)
13C CP-MAS NMR spectra and tendon (C) collagens.
763
of
dentin
(A),
LO 20 bone with a signals
decalcified
Vol.
167,
No.
BIOCHEMICAL
2, 1990
I. 13C chemical and unmineralized
Table
Tissues
Skin Tendon Demineralized bone Bone
Demineralized dentin Dentin
respectively.
The
collagen
were
collagen
helix.
Minor
in
good
significant
chemical
shifts
of
chemical
shift that
of in
than
in
the
shift peptide Pro
and
represent form.
29.3 30.5
25.5 25.3
17.6
70.1 70.7
58.9 58.9
42.7 42.9
37.6 38.6
29.5 29.1
24.9 25.2
17.5 17.6
70.7 70.5
59.2 59.3
42.8 42.5
38.8 39.6
29.7 30.2
25.2 25.1
17.6 17.5
Cp
chemical
is
the
was
the
possibility
is
that tight
Hyp
around change
the
Hyp the
can
the
Hyp residue.
(23).
residue
from
of
distortion
hydroxyproline packing 764
of
collagen.
The
Cp
a mineral
collagen
chemical of
By analogy
Hyp
bulky
Hyp
some
transition
of
with
after
be attributed
cis-trans
shift
residue
The
to
bone
tendon
signal
such
between
Even
in
Cp
with
typical
retained
in
residue
causes
molecular
still
that
bone
ppm downfield
I).
prominent
to
the
observed
a few
was
and
tissues.
(Table
less
a proline
possible
of
be
deviated
shift
to
in
the
value
tissue
known
of
the
dentin
and unmineralized
downfield
the
of
could
dentin
of
a distortion One of
shifts
with
comparable
including
by
37.2 38.7
downfield
was
interaction
distorted
42.8 43.1
change
Cp
,
58.9 59.5
shift
bonds Cp
70.8 71.1
shift
conformational Pro
Ala cp
unmineralized
dentin,
of
Pro cy
mineralized
The downfield to
Pro cp
agreement
downfield
COMMUNICATIONS
of mineralized collagens
differences
the
The
extent.
shifts tissue
RESEARCH
Pro GUY HYP ca ca cp HYP Co!,6
Hyp CB in
demineralization
BIOPHYSICAL
HYP cy
Ala
but
from
AND
is the
with
assumed typical is
(24). side
mineralized
to trans
a direct Another chain
was tissue
Vol.
167,
No.
2, 1990
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
I
60
Fiqure
3.
31P CP-MAS
spectrum was recorded 161.98 MHz.
collagen than
that
(11). of Pro
Certain also
Side
0 -20 PPm
-40
-60
NMR spectrum of decalcified dentin. with a Bruker MSL-400 spectrometer
movement
of
Hyp is
The at
more restricted
(15).
characteristics
observed
of
in carbonyl from
and bone collagen
(Figs.
not
20
chain
ppm was separated
were
40
resolved
into
dentin
carbon. the
A smaller
main signal
1,
and bone collaqens
at
As signals
2).
each amino acid,
signal
further
170-171
ppm in dentin
173-174
of
at
were
carbonyl analysis
carbons was not
attempted. Dentin
insoluble
phosphoprotein,
3).
was
confirmed
demineralized
The line
contains
phosphophoryn.
phosphoprotein completely
collagen
width
of
The by
dentin the
the
detecting
with
signal
covalently
attached
presence the
phosphate
31P solid-state
was large,
since
of
the in
NMR (Fig. the
phosphate
was bound to a macromolecule. ACKNOWLEDGMENTS The authors are grateful to the NMR laboratory, Faculty of Engineering, Hokkaido University, for NMR analysis, and to Dr. H. Saito, National Cancer Center Research Institute, for valuable scientific discussions. This study was supported by the Japanese 765
Vol.
167,
No.
BIOCHEMICAL
2, 1990
Ministry of for Scientific
Education, Research.
AND
Science
and
BIOPHYSICAL
RESEARCH
Culture
under
COMMUNICATIONS
a Grant-in-Aid
REFERENCES 1. 2.
A. (1984) In Extracellular Matrix Biochemistry (PieZ, Veis, X.A. and Reddi, A-H., Eds.), pp329-374, Elsevier, Amsterdam. Veis, A.and Schlueter, R.J. (1964) Biochemistry 3, 16501656.
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