- Email: [email protected]
A novel fluor in insoluble collagen: A crosslinking moiety in collagen molecule
Vol. 107, No. 4, 1982
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
August 31, 1982
Pages 1252-1257
A NOVEL FLUOR
IN I N S O L U B L E
COLLAGEN:
A CROSSLINKING MOIETY
IN COLLAGEN MOLECULE Tadao Ogawa,
Toshio Ono. Morizo Tsuda,
Department of Chemistry,
School of Hygienic
Kitasato 1-15-1 Kitasato, Received
June
and Yasuhiro Kawanishi Sciences,
University
Sagamihara,
Kanagawa,
228
Japan
23, 1982
A novel fluorescent compound, which was eluted slightly slower than pyridinoline on an amino acid analyzer, was obtained from acid-hydrolysate of insoluble collagen in bovine femur. Its uv absorption- and fluorescence spectra were very similar to those of pyridinoline. The results of extensive investigations of the nmr spectra in aliphatic region lead to the conclusion that it is a pyridinium derivative, tentatively named deoxypyridinoline which is very similar to pyridinoline and lacks the aliphatic hydroxyl group.
INTRODUCTION Many types of crosslinks
in collagen molecules
nated from lysyl and/or hydroxylysyl crosslinks
in pyridinoline
hydroxylysinaldehyde therefore,
formed.
Similarly,
suggested
and hydroxylysyl
a hydroxylysyl
due, an alternative
were
residue
fluorescent
residues
The
to be originated (2)(3)
from If,
is replaced with a lysyl resi-
pyridinium derivative
if only lysyl residues
should be
are concerned,
we may
(without 3-hydroxyl
derivative.
In this paper, we describe zation of a novel
in vivo.(1)
residues.
expect the formation of a non-fluorescent group) pyridinium
are origi-
fluorescent
the isolation
and characteri-
compound which supported
the above
assumption. EXPERIMENTAL Insoluble collagen bovine femur as described
Insoluble collagen was prepared in previous paper. (4)
0006-291X/82/161252-06501.00/0 Copyright © 1982 ~ A c a ~ m ~ Press, ~c. Agr~hts ~reproductionina~form reserve.
1252
from
Vol. 107, No. 4, 1982
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
o LO
o
O
o
@
A
A
A
GlyAla Fig. 1
A
A
A A
A
A
Arg
Tyr Phe Hyl LySNH3His
Amino acid analysis of the fluorescent fraction of bovine femur acid hydrolysate after a phosphocellulose column chromatography.
Pyridinoline and deoxypyridinoline Insoluble collagen was hydrolyzed in 6N(redistilled) hydrochloric acid under nitrogen at II0°C for 24 h. The hydrolysate was dried, dissolved in water and adjusted to pH 2.2 with 6M NaOH. It was applied on a phosphocellulose column pre-equilibrated with 0.2M acetate buffer(pH 2.2). After washing out ordinary amino acids using one column volume of the above buffer, the fluorescent fraction was eluted with 0.2M acetate buffer(pH3.5). Separation of the fluorescent compounds was achieved on an ion-exchange resin column (Hitachi #2612, 0.9 X I 0 cm) by the use of 0.2M succinate buffer (pH 3.25, stepwisely changed to p H 4 . 2 5 after lh) being monitored by absorption at 325 nm. Each fraction was desalted with a Biogel P-2 column (2 X 80 cm) using 0.5M acetic acid. Spectroscopic observation Nmr spectra of both compounds in D20 were observed on a Bruker CXP-300 spectrometer (300 MHz). Fluorescence and absorption spectra were observed as described in previous paper. (4) Amino acid analysis Amino acid analysis was performed using a Hitachi 835 analyzer.
RESULTS AND DISCUSSION In an attempt preparation,
to improve the efficiency
a phosphocellulose
place instead of Biogel P-2 analytical cellulose
column was used in the first
(4). Fig.
feature of the fluorescent column.
with respect
of pyridinoline
1 shows an amino acid fraction
from the phospho-
Compound A was identical with pyridinoline
to the elution characteristics,
was eluted slightly
while compound
slower than compound A. To investigate 1253
B their
Vol. 107, No. 4, 1 9 8 2
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
=E co v
c
cx~
o
Fig. 2
i
i
20
40
I
|
I
I
I
60 80 100 ]20 140 Elution volume / ml
Separation
of
compound
B from
A.
Compound B
Compound A (Pyridinoline)
I
I
I
I
I
A
I
I
8
7
6
5
4
3
2
1
Fig. 3
High resolution compound B.
IH nmr spectra of pyridinoline
1254
and
ppm
Vol. 107, No. 4, 1982
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
Y
~J
rj .r-I
r--q
0
E 0 u
r
0
:2 :~-
~
212
:~
i
~)
t~ .r.-I
E -
4J
l
r--
m
~ ~.~
r'--
i
taO
g r-
L.-~
"
".1"
~ m
-
)
1255
Vol. 107, No. 4, 1982
properties,
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
a large amount of bovine femur was decalcified,
hydrolyzed and chromatographed with phosphocellulose column. Two fluorescent compounds
(A and B, in 5:1 abundance) were then
separated by ion~exchange resin column chromatography (Fig. 2). Compound A was identified as pyridinoline by uv absorption-, fluorescence-,
and IH and 13C nmr spectrometry and ion-exchange
chromatography.
Fluorescence- and uv absorption characteristics
(including pH dependence) of compound B were also very similar to those of pyridinoline.
Titration results revealed that com-
pound B has a fluor whose pK value is about 4, just as that of pyridinoline
(4). A high resolution IH nmr spectrum of the com-
pound, however, was distinctly different from that of pyridinoline, as clearly shown in Fig. 3. An extensive investigation in the aliphatic region nmr through series of decoupling experiments allowed the complete assignments of all the signals 4). From the results and spectroscopic characteristics,
(Fig.
it can
be concluded that compound B is N-(5-amino-5-carboxy-pentyl)-3hydroxy-4-(2-amino-2-carboxy-ethyl)-5-(3-amino-3-carboxy-propyl) -pyridine. This conclusion strongly suggests that compound B, tentatively named deoxypyridinoline,
results from inter- and/or
intra crosslinking of collagen molecules. Recently, Deyl et al.
(5), in their investigation on
pyridinoline content in elastin, evidence,
suggested, without substantial
the existence of fluorescent crosslinking substance
other than pyridinoline.
It is thus probable that deoxypyridino-
line exists also in elastin.
ACKNOWLEDGMENTS We thank Drs. H. Saito and M. Maeda, National Cancer Center Research Institute,
for their assistance in nmr spectra and for
helpful discussions. 1256
Vol. 107, No. 4, 1 9 8 2
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES I. Bornstein, P. and Traub, W., in "The Proteins", Vol. 4, 3rd ed., pp 411 - 632 (eds. Neurath, H. and Hill, R. L., Academic Press, New York, 1979) 2. Fujimoto, D., Moriguchi, T., Ishida, T., and Hayashi, H., Biochem. Biophys. Res. Commun., 84, 52 - 57. (1978) 3. Eyre, D. R., and Oguchi, H., ibi~., __92, 403 - 410 (1980) 4. Tsuda, M., Ono, T., Ogawa, T., and Kawanishi, Y., ibid,., 104, 1407 - 1412. (1982) 5. Deyl, Z., Horakova, M., and Vancikova, 0., Mech. Ageing Dev., 17, 321 - 325. (1981)
1257
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
August 31, 1982
Pages 1252-1257
A NOVEL FLUOR
IN I N S O L U B L E
COLLAGEN:
A CROSSLINKING MOIETY
IN COLLAGEN MOLECULE Tadao Ogawa,
Toshio Ono. Morizo Tsuda,
Department of Chemistry,
School of Hygienic
Kitasato 1-15-1 Kitasato, Received
June
and Yasuhiro Kawanishi Sciences,
University
Sagamihara,
Kanagawa,
228
Japan
23, 1982
A novel fluorescent compound, which was eluted slightly slower than pyridinoline on an amino acid analyzer, was obtained from acid-hydrolysate of insoluble collagen in bovine femur. Its uv absorption- and fluorescence spectra were very similar to those of pyridinoline. The results of extensive investigations of the nmr spectra in aliphatic region lead to the conclusion that it is a pyridinium derivative, tentatively named deoxypyridinoline which is very similar to pyridinoline and lacks the aliphatic hydroxyl group.
INTRODUCTION Many types of crosslinks
in collagen molecules
nated from lysyl and/or hydroxylysyl crosslinks
in pyridinoline
hydroxylysinaldehyde therefore,
formed.
Similarly,
suggested
and hydroxylysyl
a hydroxylysyl
due, an alternative
were
residue
fluorescent
residues
The
to be originated (2)(3)
from If,
is replaced with a lysyl resi-
pyridinium derivative
if only lysyl residues
should be
are concerned,
we may
(without 3-hydroxyl
derivative.
In this paper, we describe zation of a novel
in vivo.(1)
residues.
expect the formation of a non-fluorescent group) pyridinium
are origi-
fluorescent
the isolation
and characteri-
compound which supported
the above
assumption. EXPERIMENTAL Insoluble collagen bovine femur as described
Insoluble collagen was prepared in previous paper. (4)
0006-291X/82/161252-06501.00/0 Copyright © 1982 ~ A c a ~ m ~ Press, ~c. Agr~hts ~reproductionina~form reserve.
1252
from
Vol. 107, No. 4, 1982
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
o LO
o
O
o
@
A
A
A
GlyAla Fig. 1
A
A
A A
A
A
Arg
Tyr Phe Hyl LySNH3His
Amino acid analysis of the fluorescent fraction of bovine femur acid hydrolysate after a phosphocellulose column chromatography.
Pyridinoline and deoxypyridinoline Insoluble collagen was hydrolyzed in 6N(redistilled) hydrochloric acid under nitrogen at II0°C for 24 h. The hydrolysate was dried, dissolved in water and adjusted to pH 2.2 with 6M NaOH. It was applied on a phosphocellulose column pre-equilibrated with 0.2M acetate buffer(pH 2.2). After washing out ordinary amino acids using one column volume of the above buffer, the fluorescent fraction was eluted with 0.2M acetate buffer(pH3.5). Separation of the fluorescent compounds was achieved on an ion-exchange resin column (Hitachi #2612, 0.9 X I 0 cm) by the use of 0.2M succinate buffer (pH 3.25, stepwisely changed to p H 4 . 2 5 after lh) being monitored by absorption at 325 nm. Each fraction was desalted with a Biogel P-2 column (2 X 80 cm) using 0.5M acetic acid. Spectroscopic observation Nmr spectra of both compounds in D20 were observed on a Bruker CXP-300 spectrometer (300 MHz). Fluorescence and absorption spectra were observed as described in previous paper. (4) Amino acid analysis Amino acid analysis was performed using a Hitachi 835 analyzer.
RESULTS AND DISCUSSION In an attempt preparation,
to improve the efficiency
a phosphocellulose
place instead of Biogel P-2 analytical cellulose
column was used in the first
(4). Fig.
feature of the fluorescent column.
with respect
of pyridinoline
1 shows an amino acid fraction
from the phospho-
Compound A was identical with pyridinoline
to the elution characteristics,
was eluted slightly
while compound
slower than compound A. To investigate 1253
B their
Vol. 107, No. 4, 1 9 8 2
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
=E co v
c
cx~
o
Fig. 2
i
i
20
40
I
|
I
I
I
60 80 100 ]20 140 Elution volume / ml
Separation
of
compound
B from
A.
Compound B
Compound A (Pyridinoline)
I
I
I
I
I
A
I
I
8
7
6
5
4
3
2
1
Fig. 3
High resolution compound B.
IH nmr spectra of pyridinoline
1254
and
ppm
Vol. 107, No. 4, 1982
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
Y
~J
rj .r-I
r--q
0
E 0 u
r
0
:2 :~-
~
212
:~
i
~)
t~ .r.-I
E -
4J
l
r--
m
~ ~.~
r'--
i
taO
g r-
L.-~
"
".1"
~ m
-
)
1255
Vol. 107, No. 4, 1982
properties,
BIOCHEMICAL A N D BIOPHYSICAL RESEARCH COMMUNICATIONS
a large amount of bovine femur was decalcified,
hydrolyzed and chromatographed with phosphocellulose column. Two fluorescent compounds
(A and B, in 5:1 abundance) were then
separated by ion~exchange resin column chromatography (Fig. 2). Compound A was identified as pyridinoline by uv absorption-, fluorescence-,
and IH and 13C nmr spectrometry and ion-exchange
chromatography.
Fluorescence- and uv absorption characteristics
(including pH dependence) of compound B were also very similar to those of pyridinoline.
Titration results revealed that com-
pound B has a fluor whose pK value is about 4, just as that of pyridinoline
(4). A high resolution IH nmr spectrum of the com-
pound, however, was distinctly different from that of pyridinoline, as clearly shown in Fig. 3. An extensive investigation in the aliphatic region nmr through series of decoupling experiments allowed the complete assignments of all the signals 4). From the results and spectroscopic characteristics,
(Fig.
it can
be concluded that compound B is N-(5-amino-5-carboxy-pentyl)-3hydroxy-4-(2-amino-2-carboxy-ethyl)-5-(3-amino-3-carboxy-propyl) -pyridine. This conclusion strongly suggests that compound B, tentatively named deoxypyridinoline,
results from inter- and/or
intra crosslinking of collagen molecules. Recently, Deyl et al.
(5), in their investigation on
pyridinoline content in elastin, evidence,
suggested, without substantial
the existence of fluorescent crosslinking substance
other than pyridinoline.
It is thus probable that deoxypyridino-
line exists also in elastin.
ACKNOWLEDGMENTS We thank Drs. H. Saito and M. Maeda, National Cancer Center Research Institute,
for their assistance in nmr spectra and for
helpful discussions. 1256
Vol. 107, No. 4, 1 9 8 2
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES I. Bornstein, P. and Traub, W., in "The Proteins", Vol. 4, 3rd ed., pp 411 - 632 (eds. Neurath, H. and Hill, R. L., Academic Press, New York, 1979) 2. Fujimoto, D., Moriguchi, T., Ishida, T., and Hayashi, H., Biochem. Biophys. Res. Commun., 84, 52 - 57. (1978) 3. Eyre, D. R., and Oguchi, H., ibi~., __92, 403 - 410 (1980) 4. Tsuda, M., Ono, T., Ogawa, T., and Kawanishi, Y., ibid,., 104, 1407 - 1412. (1982) 5. Deyl, Z., Horakova, M., and Vancikova, 0., Mech. Ageing Dev., 17, 321 - 325. (1981)
1257