From the Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, United Kingdom
Received for publication, October 18, 2000, and in revised form, February 15, 2001
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ABSTRACT |
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The structures of pyrrolic forms of cross-links
in collagen have been confirmed by reacting collagen peptides with a
biotinylated Ehrlich's reagent. This reagent was synthesized by
converting the cyano group of
N-methyl-N-cyanoethyl-4-aminobenzaldehyde to a
carboxylic acid, followed by conjugation with biotin pentyl-amine. Derivatization of peptides from bone collagen both stabilized the
pyrroles and facilitated selective isolation of the pyrrole-containing peptides using a monomeric avidin column. Reactivity of the
biotinylated reagent with collagen peptides was similar to that of the
standard Ehrlich reagent, but heat denaturation of the tissue before
enzyme digestion resulted in the loss of about 50% of the pyrrole
cross-links. Identification of a series of peptides by mass
spectrometry confirmed the presence of derivatized pyrrole structures
combined with between 1 and 16 amino acid residues. Almost all of the
pyrrole-containing peptides appeared to be derived from N-terminal
telopeptide sequences, and the nonhydroxylated (lysine-derived) form
predominated over pyrrole cross-links derived from helical hydroxylysine.
The chemistry of the lysine-derived collagen cross-links is
relatively complex, with collagen type, tissue location, and age of the
protein all influencing the range of cross-links present (1). In bone,
cartilage, and tendon, extensive hydroxylation of telopeptide lysine
residues leads to the formation of hydroxylysyl aldehydes. These
aldehydes interact with the Isolation and characterization of the pyrrolic cross-link(s) have been
hampered by the instability of the pyrrole to acid or alkali
hydrolysis. Enzyme digests of decalcified bone matrix have been used in
attempts to isolate pyrrole-containing peptides, but because these
peptides are reduced in size and enriched, the pyrrole tends to oxidize
or polymerize.1 In a recent
study, Ehrlich chromogen-containing peptides were enriched and analyzed
by mass spectrometry, revealing peaks that were interpreted to be
derived from peptides containing oxidized pyrrolic cross-link (11).
These experiments supported previous preliminary indications that the
pyrroles were concentrated at the N-telopeptide site. Estimation of the
pyrrole has been made using the color yield of digests with Ehrlich's
reagent and correlated to the strength of tendon (12). The content of
pyrrole decreases with age in mature animals (13, 14) and is also
lowered in mineralizing tissue (15) and the osteoporotic femoral head
compared with normal (16).
The aim of the present study was to confirm the proposed pyrrole
structures by isolating and fully characterizing small peptides containing pyrrolic cross-links. To minimize degradation of pyrroles, the collagen peptides were derivatized and stabilized by reaction with
a synthetic, biotinylated Ehrlich's reagent designed to facilitate rapid isolation of cross-linked peptides using a monomeric avidin column. The resulting peptides were separated by
HPLC2 and analyzed by mass spectrometry.
Preparation of Biotinylated Ehrlich's Reagent
The most appropriate starting material for the synthesis,
N-methyl-N-cyanoethyl-4-aminobenzaldehyde
(I; Fig. 1), was a gift from
Dr. Huang XuGuang (Eternwin Chemicals Ltd, Beijing, China).
After conversion of the nitrile to a carboxylic acid group, the latter
was conjugated to biotin pentyl-amine via a carbodiimide reaction.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
amino group of lysine or hydroxylysine
residues from the adjacent helix to initially form Schiff bases that
can chemically rearrange to form a more stable, keto-imine difunctional
cross-link. In skin, the telopeptides are not hydroxylated, and no such
rearrangement of the Schiff base cross-link occurs. These two different
chemistries, dependent on telopeptide lysine hydroxylation, lead to
the formation of different mature cross-links by further reaction of
the difunctional cross-links in a tissue-specific manner: hydroxylation
of the telopeptide lysine residues appears to be accomplished by
tissue-specific enzymes, distinct from those that hydroxylate lysines
in the helix (2). For the hydroxylysyl aldehyde pathway (bone,
cartilage, and tendon), the difunctional cross-links can combine with
another hydroxylysine aldehyde-derived component to form a pyridinium cross-link. These cross-links have been extensively characterized, and
mechanisms of formation have been proposed (3, 4). However, many
studies of cross-linked collagen peptides have implied the presence of
other trifunctional cross-links because of a lack of stoichiometric
amounts of the pyridinium compounds (5, 6). The possible presence of
pyrrolic components in collagen arose from the observation that
reaction of bone with p-dimethylaminobenzaldehyde gave a
pink color characteristic of pyrroles (7); these reactive species were
named Ehrlich chromogens. Later experiments used diazo-affinity columns
(also consistent with a pyrrolic structure) to covalently bind the
Ehrlich chromogen-containing peptides from enzyme digests of bone (8)
and skin (9) and show by amino acid analysis that they were derived
from collagen. A similar affinity chromatography approach was used to
demonstrate that Ehrlich chromogen cross-links were present at the same
loci as the pyridinium cross-links in bovine tendon (10). This work culminated in a proposed structure and mechanism of formation for
pyrroles analogous to that for pyridinium cross-link formation: this
mechanism involves reaction of a difunctional, keto-imine cross-link
with a lysyl aldehyde-derived component rather than a hydroxylysyl
aldehyde-derived component (10), where the latter may be a second
difunctional cross-link (11). Taken together, these studies suggest
that Ehrlich chromogens exist in many different tissues and probably
have different chemistries based on their stability and the different
spectra observed upon reaction with p-dimethylaminobenzaldehyde (9). The mechanism proposed for Ehrlich chromogen cross-link formation, based on work done in tendon,
is only applicable to tissues that partially utilize the hydroxylysine
aldehyde pathway: these cross-links would be expected to be absent from
skin, which lacks hydroxylysine aldehyde, and from cartilage, in which
telopeptide lysines are fully hydroxylated.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Chemical synthesis of the biotinylated
Ehrlich's reagent and HPLC fractionation of the products. The
nitrile obtained as starting material (compound I) was
hydrolyzed to the acid (compound II), and the product was
conjugated with a biotin derivative to yield the reagent (compound
III). Intermediates were purified by reversed phase HPLC
with an acetonitrile gradient monitoring the absorbance at 330 nm, and
the structures shown were confirmed by mass spectrometry.
Hydrolysis of the Nitrile to the Carboxylic Acid--
Conversion
of the cyano group (I; Fig. 1) to the corresponding acid
(II; Fig. 1) was achieved by alkali hydrolysis. The nitrile
(150 mg) was dissolved in 5 M NaOH and 6%
H2O2 (5 ml) and refluxed for 2 h. The
hydrolysate was then acidified by the addition of concentrated HCl,
dried down under vacuum, and redissolved in ethanol (1.5 ml). An
aliquot of this solution (1 ml) was added to 0.2 M NaOH (1 ml) and applied to an anion exchange column (Bio-Rad AG 1-X8;
Na+-form; 2 ml). The column was washed with water (12 ml)
before elution of the bound material with 2 M HCl. The
eluent was dried under vacuum, and the residue was resuspended in water
(1 ml). A small amount of residue (soluble in ethanol but containing no compound II) was removed, after which the aqueous fraction was dried under vacuum (yielding 7 mg of material) and redissolved in
0.1% trifluoroacetic acid (1 ml). Aliquots (100 µl) of this material
were then chromatographed on a Waters RCM Prep-Pak® C18 column (25 × 100 mm; 10 µm) pumped at 4 ml
min1. The buffers used were 0.1%
trifluoroacetic acid (buffer A) and 70% acetonitrile, 0.1%
trifluoroacetic acid (buffer B) with a gradient of 5 min at 5% buffer
B followed by a linear increase to 70% buffer B over 35 min.
Monitoring at 330 nm showed a single major peak that eluted at 28.3 min, and fractions corresponding to this peak were pooled and dried
under vacuum (yield = 3 mg). Analysis of this material by
electrospray mass spectrometry in negative-ion mode using a MAT 900 mass spectrometer (Finnigan MAT, Bremen, Germany) revealed the major
ion as [M-H] = 206.2, which corresponds to the expected value for
N-methyl-N-proprionic acid-4-amino benzaldehyde
(Mr = 207.2; II in Fig. 1).
Biotinylation of the Carboxylic Acid Derivative-- N-Methyl-N-proprionic acid-4-amino benzaldehyde (3 mg) was redissolved in water (3 ml), and biotin pentyl-amine (30 mg; Pierce) was added. A solution of 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide/N-hydroxysuccinimide (0.035 M/0.028 M, respectively; 3 ml) was added and heated to 50 °C for 4 h. The resulting solution was then dried down under vacuum and chromatographed using the preparative RCM Prep-Pak® column as described previously. The gradient applied was 20% buffer B for 5 min followed by a linear increase to 60% buffer B over 30 min. The chromatogram (Fig. 1) showed two major peaks, with the peak eluting at 15 min corresponding to the unreacted acid. The second major component eluting at 18 min was analyzed by positive-ion electrospray mass spectrometry and showed [M+H] of 518.7 and [M+Na] of 540.6, corresponding to the calculated Mr of 517.7
Preparation of Bone Peptides
De-fatted human bone (20 g) was powdered in a Spex freezer-mill in liquid nitrogen. The resultant powder was decalcified by three 2-day extractions in 0.5 M EDTA, pH 8, at 4 °C, washed with water, and lyophilized. The decalcified bone was then digested using the following enzymic procedures.
Trypsin-- The bone powder (1.0 g) suspended in 50 mM ammonium bicarbonate, pH 8, was denatured by heating at 70 °C for 30 min, and after cooling to 37 °C, trypsin (Sigma) was added to give an enzyme substrate ratio of 1:30. The mixture was incubated with gentle agitation for 18 h at 37 °C, followed by centrifugation of the mixture and lyophilization of the supernatant solution. The proportion of collagen solubilized was determined by hydroxyproline analysis.
Cathepsin K-- Portions (1.0 mg) of bone powder were suspended in 200 µl of 50 mM sodium acetate buffer, pH 5.0, containing 2 mM EDTA and 2 mM dithiothreitol. One sample was heat-denatured by heating at 70 °C for 30 min and then cooled to 37 °C. Ten µg of recombinant human cathepsin K (a gift from SmithKline Beecham Pharmaceuticals, King of Prussia, PA) in 12.5 µl of PBS was added to both samples. After a 24-h incubation at 37 °C with gentle agitation, the same amount of cathepsin K was added, and the digestion was continued for an additional 24 h. After centrifugation (13,000 × g) of any undigested tissue, an aliquot (20 µl) of the supernatant was taken for hydroxyproline determination, and the remainder was used directly for reaction with Ehrlich reagents.
Papain/Protease X-- The decalcified bone powder (1.1 g) was suspended in 0.1 M citrate buffer, pH 5, heated to 70 °C for 1 h to denature the triple-helical structure, and allowed to cool to 45 °C. Papain (100 units) was added, and the digest was incubated for 4 h. The pH of the digest was then adjusted to pH 7.4 by the addition of 2 M Tris, and the temperature was lowered to 37 °C for an overnight digestion with protease type X (100 units). The completed digest (estimated as 110 µM collagen by total pyridinium cross-link content) was frozen, lyophilized, and suspended in water (7 ml).
Reaction with Ehrlich Reagents
For comparisons of the reactivity of trypsin and cathepsin K
digests of bone, the peptide digest (140 µl) containing up to 25 mM hydroxyproline was mixed with 27 µl of 70 mM Ehrlich's reagent (10 mg ml1)
or 70 mM biotinylated Ehrlich's reagent (36 mg
ml
1) dissolved in 2-methoxyethanol, followed
immediately by 83 µl of 12 M HCl. After 30 min at room
temperature, the color was determined spectrophotometrically.
The papain/protease X bone digest (500 µl) was acidified by the addition of 12 M HCl (250 µl) before adding biotinylated Ehrlich's reagent (50 µg). After 30 min at room temperature, the acid was neutralized by the addition of 12 M NaOH (approximately 220 µl) followed by the addition of 40 mM phosphate buffer (20 ml).
Isolation of Biotinylated, Pyrrolic Peptides Using a Monomeric Avidin Column
A monomeric avidin column (5 ml) was prepared according to the
manufacturer's (Pierce) instructions. The reacted bone digest at
neutral pH was added slowly to the column, which was then washed with 6 column volumes of PBS followed by 1 column volume of water. The
biotinylated material was eluted at about 1 ml
min1 with 1 M acetic acid
adjusted to pH 2.5 with ammonia, and 8 fractions (5 ml) were collected.
Estimation of Biotinylated Compounds by Competitive Enzyme-linked Assay-- To assess the efficiency of the monomeric avidin column, a competitive enzyme-linked immunosorbent assay was developed. Immulon 4 immunnoassay plates were coated with streptavidin (25 nM) in PBS for 2 h at 37 °C. Samples or standards in PBS/0.1% Tween and 0.5% fat-free milk powder (110 µl) were added to biotinylated peroxidase (Sigma; 10 ng/ml; 110 µl) in PBS/Tween and 0.5% fat-free milk powder in a U-bottomed 96-well plate. The mixed samples were then transferred to the washed, streptavidin-coated plate and incubated for 90 min at 37 °C. After washing the plate three times with PBS/0.1% Tween, the peroxidase substrate (200 µl) tetramethyl-benzidine dihydrochloride was added (0.1 mg/ml) in 0.05 M citrate/phosphate buffer, pH 5, 0.012% v/v hydrogen peroxide. The reaction was stopped after 15 min by the addition of 3 M sulfuric acid (50 µl).
Analysis of Isolated Material by HPLC--
Material eluted from
the avidin column was reduced in volume (100 µl) and chromatographed
on a reversed phase HPLC column (4.6 × 100 mm; C18;
particle size, 3 µm). The column was equilibrated with 0.1%
trifluoroacetic acid (buffer A), and peptides were eluted over 35 min
with linear gradients formed with 70% acetonitrile and 0.1%
trifluoroacetic acid (buffer B). The eluant was monitored at 214, 280, and 330 nm. Each fraction from the HPLC was dried down and redissolved
in water (2 µl). An aliquot (1 µl) was mixed with
-cyano-4-hydroxy-cinnamic acid (1 µl of a 10 mg/ml solution in
70% acetonitrile and 0.1% trifluoroacetic acid), dried onto a sample
plate, and analyzed by MALDI-TOF mass spectrometry (Voyager-DE; Applied
Biosystems) calibrated externally using bradykinin.
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RESULTS |
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Hydroxyproline analysis showed that ~75% of the heat-denatured
bone had been solubilized by trypsin digestion. For cathepsin K, the
bone was completely digested after heat denaturation, whereas only 45%
of the collagen was solubilized without heat treatment. Comparison of
the reactivity of the tryptic peptides with Ehrlich reagent and
biotinylated Ehrlich reagent showed an apparently enhanced color yield
with the biotinylated derivative, with a slight shift in absorption
maximum to 579 nm (Fig. 2). Under the conditions of reaction, Ehrlich's reagent showed a linear response in
color development between 1 and 25 mg ml1
collagen peptides. For the cathepsin K-derived peptides, similar color
development at 579 nm was observed despite the differences in peptide
concentration. Thus, the heat-denatured sample (4.5 mg
ml
1 collagen) had A579 = 0.024, whereas the sample solubilized without heat denaturation (1.9 mg ml
1) showed A579 = 0.021: the two spectra were similar (Fig. 2).
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After reaction of bone-derived peptides with the biotinylated reagent
and selection of derivatized pyrrole-containing components by
adsorption to a monomeric avidin column, the total amino acid content of the material eluted from the column was determined (Table
I). The composition was consistent with a
preparation rich in telopeptide material, with relatively high
proportions of Ser, Ala, Asp, Glu, Thr, and Ile and low amounts of Pro
and hydroxyproline. The pattern of elution from the monomeric
avidin column, as measured by the biotin inhibition assay (Fig.
3), showed that the biotinylated material
was not easily displaced from the column. Indeed, previous experiments
had shown that a fast flow-rate and large volumes of acetic acid (or 2 mM biotin, as suggested by the manufacturer) were required
to displace the biotin derivatives from the column. The reversed phase
HPLC profile of the eluted material was dominated by peaks eluting at
21 min (representing free biotin) and at 34 min (comprising unreacted
biotinylated Ehrlich's reagent); the former probably arises from
continuous "leeching" from the column of biotin used in its
preparation to block high affinity sites. Many of the pyrrole
cross-link-containing peptides were not detectable monitoring the
column at the lowest feasible wavelength (214 nm) because of their
small size and the relatively small amounts present. Each fraction was
therefore analyzed by MALDI-TOF mass spectrometry, yielding the spectra shown in Fig. 4. Because there were
insufficient quantities of many of the smaller peptides to obtain amino
acid composition data, some ambiguities in their structural assignments
did arise. In particular, the mass difference between Glu and Ile/Leu
is equivalent to an additional hydroxyl group, and for the isolated peptide with Mr = 1086 (Fig. 4a), the
ambiguity is due to the possible presence of a hydroxylated cross-link.
Thus, this peptide may contain Gly and Glu (from either the C- or
N-telopeptides of the 1 chain) or, for a hydroxylated cross-link, a
Gly residue linked with either Ile (from the
1 helix) or Leu (from
the
2 helix). Even when the amino acid composition is known, the
precise location of the residues may not be clear, as in the case of
the peptide with Mr = 957 (Fig. 4a)
containing the biotinylated pyrrole with a single Gly residue. This
residue is shown in a helical position (which could be at the N- or
C-terminal overlap sites) but could also be derived from the
2(I)
N-telopeptide: this peak may contain a mixture of Gly-containing
peptides from the different locations. The Mr = 1029 peptide shown in Fig. 4f could have the same
alternatives of glutamate or hydroxylated pyrrole-leucine/isoleucine. The peaks corresponding to a loss of Gly (Fig. 4, b and
c) are probably losses due to the energy of the laser
desorption rather than discrete peptides, but these peaks provide
additional evidence for the peptide structures proposed. The structures
of the larger peptides shown in the other panels are unambiguous.
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Except for the large peptide in Fig. 4h, the peptides
described here could all originate from the N-telopeptide site,
although the smaller peptides could originate from either the N- or
C-terminal site. The peptides isolated and identified by MALDI-TOF mass
spectrometry were broadly consistent with the amino acid composition of
the total derivatized pyrrole cross-link fraction, although the spectra give little information on the relative quantities of the peptides. However, the presence of a relatively large proportion of alanine in
the amino acid composition of the total cross-link fraction (Table I)
indicates that not all of the pyrrole-containing peptides have yet been
accounted for by the MALDI-TOF analysis.
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DISCUSSION |
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The results presented show that Ehrlich's reagent can be coupled to biotin via the tertiary amine group and maintain its ability to react with a model pyrrole giving the characteristic color development. A similar color reaction, as confirmed by the absorption spectrum, was observed after reaction with several different enzymic digests of bone peptides. The isolation and identification of a series of peptides from enzyme digests of human bone collagen provide direct confirmation of the structure of the pyrrolic cross-links. Previous studies in which pyrrole-containing peptides were identified after isolation from digests of bovine tendon using diazotization methods (10) did not identify the cross-linking compounds.
Taking into account the differences in peptide concentration, the
reactivity at 579 nm of the cathepsin K digest after heat denaturation
(5.3 units mg1) was similar to that for the
tryptic digest of heat-denatured bone (5.6 units
mg
1). However, reactivity of the bone digest
solubilized by cathepsin K without heat treatment was about 2-fold
higher (11.2 units mg
1), suggesting that
around 50% of the pyrrole cross-links in bone collagen may be unstable
to the thermal denaturation step: the similarity in spectra of the
biotinylated Ehrlich reaction products indicates that similar
cross-linking moieties are being detected. For future quantitative
studies of the location of pyrroles within the collagen fibril, this
susceptibility to degradation must be taken into account. The primary
objective of the present study, however, was to characterize the
structure of the pyrrolic cross-links, for which quantitative recovery
was not essential.
In isolating material from the papain/protease X digest of bone, a
monomeric avidin column proved effective in binding biotinylated pyrrole peptides, although a large volume of acid was needed to elute
the bound material. The results obtained from MALDI-TOF analyses of the
isolated peptides after separation by reversed phase HPLC were
interpreted in light of the known sequences in the region of the
cross-linking sites involving the N- and C-terminal locations, as shown
in Fig. 5. These calculations also took
into account that the complexes formed by reaction of the pyrrolic cross-links with biotinylated Ehrlich's reagent, stabilized through delocalization of the charge as shown in Fig.
6, would contribute relative masses of
899 and 915 for the lysine- and hydroxylysine-derived cross-links, respectively. Peptides from the N-terminal cross-linking site can comprise two 1(I) telopeptides or an
1(I) telopeptide cross-linked to an
2(I) telopeptide. Either of these combinations can cross-link with the helical region from either chain. At the C-terminal cross-linking site, only the
1(I) chain contains a cross-link site, which can cross-link with either chain in the helix.
As indicated in Fig. 4, it is likely that the pyrrole cross-link will
be present in both a lysine and hydroxylysine form, depending on the
hydroxylation state of the helical lysine contributing to the
cross-link.
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The primary purpose of the present study was to generate small peptides that would, in conjunction with the novel derivatization method, allow accurate assessment of the proposed cross-link structure. This has been achieved as the observed masses of a relatively large number of isolated peptides were extremely close to the theoretic masses of derivatized complexes at the N- and C-telopeptide sites. These data, together with the limited peptide sequencing information through ladder fragmentation, provide strong confirmatory evidence for the proposed structure of the pyrrolic cross-link (10). However, speculation on the distribution of the pyrrole within the molecule should be treated with caution because of ambiguities in the assigned structures of the smaller peptides and the nonspecific nature of the proteolysis. Consistent with previous studies that indicate that the pyrrolic cross-links are located mainly at the N-terminal sites (10, 11), almost all of the isolated peptides could have originated from N-telopeptide-related sequences. However, one of the larger peptides (see Fig. 4h) was clearly derived from the C-terminal telopeptide, and it is conceivable that the nonspecific proteolytic digestion conditions may have cleaved C-terminal components further to the peptides observed in Fig. 4, a, b, and f. The presence of relatively high proportions of Ala in the amino acid digest is also indicative of C-telopeptide-derived pyrrole not yet detected by MALDI-TOF analysis.
Of those peptides analyzed in the present study, there appears to be a
predominance of 1N to
1N-telopeptide
cross-linked through the pyrroles: this finding is contrary to those of
Hanson and Eyre (11), who found mostly
1 to
2 telopeptide
cross-links. These differences could be accounted for by the
selectivity of both methodologies, and a more controlled proteolysis
followed by analysis of all the pyrrole-containing peptides produced is
required to definitively establish the distribution of pyrrole
cross-links. As for the pyridinium cross-links, hydroxylation of the
helical lysine will give rise to different forms of the pyrrole, and
the analyses show that both hydroxylated and nonhydroxylated forms are
present. Consistent with previous trivial names, the nomenclature
pyrrololine (PYL) and deoxypyrrololine (DPL) has been proposed for
these cross-links (17). The presence of the homologues is most
clearly evident in the peptides identified in Fig. 4, d and
e, which appear to be hydroxylation isoforms of the same
peptide. However, the unhydroxylated form appears to predominate,
consistent with a relatively higher proportion of the lysyl form of
pyridinium cross-link at the N-telopeptide site. No evidence was
obtained for glycosylated derivatives of the hydroxylysyl form of
pyrrole cross-link, although these might be expected to occur,
particularly involving the C-telopeptide cross-link.
In conclusion, this study provides strong evidence for the structures
of pyrrolic cross-links in collagen proposed by Kuypers et
al. (10). The results suggest that a hydroxylated form of the
pyrrole exists, analogous to the formation of pyridinoline and
deoxypyridinoline. The pyrrole appears to occur predominantly at the
N-telopeptide site, although additional studies using a more consistent
digestion system will be needed to determine the concentrations,
intramolecular distribution, and tissue specificity of these
potentially important cross-links.
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ACKNOWLEDGEMENTS |
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We thank Dr. Maxine Gowen (SmithKline Beecham Pharmaceuticals) for providing recombinant human cathepsin K and Phyllis Nicol for skillful technical assistance.
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FOOTNOTES |
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* This work was supported by Metra Biosystems Inc and the Scottish Executive Rural Affairs Department.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Axis-Shield Diagnostics Limited, The Technology
Park, Dundee DD2 1XA, Scotland, United Kingdom.
§ To whom correspondence should be addressed: Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, Scotland, United Kingdom. Tel.: 44-1224-716639; Fax: 44-1224-716687; E-mail: s.robins@rri.sari.ac.uk.
Published, JBC Papers in Press, March 14, 2001, DOI 10.1074/jbc.M009506200
1 J. D. Brady and S. P. Robins, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: HPLC, high pressure liquid chromatography; PBS, phosphate-buffered saline; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight mass spectrometry.
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