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Originally published In Press as doi:10.1074/jbc.M002346200 on July 11, 2000
J. Biol. Chem., Vol. 275, Issue 42, 33014-33020, October 20, 2000
Identification of an Intermediate State in the Helix-Coil
Degradation of Collagen by Ultraviolet Light*
Christopher A.
Miles §,
Alina
Sionkowska¶,
Sarah L.
Hulin,
Trevor J.
Sims,
Nicholas C.
Avery, and
Allen J.
Bailey
From the University of Bristol, Collagen Research
Group, School of Veterinary Science, Langford, Bristol BS40 5DU, United
Kingdom and the ¶ Department of Chemistry, N. Copernicus
University, 87-100 Torun, Poland
Received for publication, March 21, 2000, and in revised form, July 5, 2000
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ABSTRACT |
Differential scanning calorimetry has
revealed the presence of a new denaturation endotherm at 32 °C
following UV irradiation of collagen, compared with 39 °C for the
native triple helix. Kinetic analyses showed that the new peak was a
previously unknown intermediate state in the collagen helix-coil
transition induced by UV light, and at least 80% of the total collagen
was transformed to random chains via this state. Its rate of formation
was increased by hydrogen peroxide and inhibited by free radical
scavengers. SDS-polyacrylamide gels showed evidence of competing
reactions of cross-linking and random primary chain scission. The
cross-linking was evident from initial gelling of the collagen
solution, but there was no evidence for a dityrosine cross-link.
Primary chain scission was confirmed by end group analysis using
fluorescamine. Electron microscopy showed that the segment long
spacing crystallites formed from the intermediate state were
identical to the native molecules. Clearly, collagen can undergo quite
extensive damage by cleavage of peptide bonds without disorganizing the
triple helical structure. This leads to the formation of a damaged
intermediate state prior to degradation of the molecules to short
random chains.
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INTRODUCTION |
Studies of the effect of UV radiation on the properties of the
collagen molecule, in solution or in its aggregated fiber form, are
rather limited. It has been reported that cross-linking and degradation
(1) occur on exposure to UV, the relative proportions depending on the
presence of oxygen, pH of solution, type of collagen, and wavelength of
the UV.
These effects have been attributed to absorption by the aromatic
groups, phenylalanine and tyrosine, with the suggestion that cross-linking could be mediated through dityrosine cross-links (2),
although no detailed chemistry has been carried out to demonstrate the
presence of this cross-link. For example, Kato et al. (3)
reported loss of tyrosine and cross-linking in both type I and IV
collagens but could not detect dityrosine, only DOPA.1 Kaminska and
Sionkowska (4) demonstrated that the infrared amide bands were shifted
to a lower frequency, indicating that structural changes were taking
place in the molecule. They also deduced that helix-random coil
transitions were taking place by reduction in the viscosity (5). Much
earlier, Bailey (6) had shown that ionizing-radiation (cobalt 60)
reduced the denaturation temperature of collagen solutions in a
biphasic manner, and Hayashi et al. (7) reported a similar
biphasic phenomenon when collagen was irradiated with UV light during
CD measurements.
The collagen family of proteins (currently 20) constitute 25% of the
total protein mass of the body and determine architecture, tissue
strength, and cell-collagen interactions. A characteristic feature of
collagen is the triple helical structure of three left-handed polyproline type helices twisted into a right-handed superhelix. The
formation of such a structure is due to the repeating sequence Gly-X-Y, where X and Y are
often proline and hydroxyproline, respectively, and hydrogen bonding
takes place between chains within the triple helix.
On heating, the triple helix unfolds to produce random chains of
gelatin (8), and this transition has been studied by polarimetry (9),
spectrometry (10), circular dichroism (7), and viscosity (11). However,
we believe that the most powerful technique is differential scanning
calorimetry (DSC), which provides direct measurements of thermodynamic
characteristics and, unlike the other methods, is equally applicable to
collagen solutions (12), fibers (13), and tissues (14). Collagen
monomers in solution unfold abruptly to yield a single, sharp, and
highly energetic endotherm. We have shown that the thermal helix-coil
transition proceeds via a single, first-order rate process (15) in
which there is no intermediate state.
In this paper, we report that the UV-induced transition proceeds via a
previously unknown intermediate state and discuss the possible
mechanisms involved. It was possible to demonstrate minor changes in
the structure of the collagen molecule resulting in the intermediate
state and follow the progression of cross-linking and degradation. An
understanding of the mechanisms involved in these changes and their
inhibition is obviously of considerable interest to the effect of UV on
the aging of dermal collagen and the use of UV in the treatment of
psoriasis, where there is a potential for damage to the underlying
collagen fibers.
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EXPERIMENTAL PROCEDURES |
Collagen Solutions
Collagen solutions were prepared from tendons freshly dissected
from frozen rat tails, dissolved in 0.5 M acetic acid, and centrifuged at 10,000 rpm for 20 min to remove undissolved material. The solution was scanned by DSC to determine the enthalpy of the transition per unit volume of the solution, and the concentration of
collagen was calculated using a transition enthalpy of 70 J/g (12). On
this basis, the solution concentration was adjusted to 2 mg/ml by the
addition of 0.5 M acetic acid, prior to UV irradiation.
Ultraviolet Irradiation
Solutions were irradiated using UV-C light from two 6-watt
fluorescent lamps (type TUV 6W; Philips, Croydon, Surrey, UK) each specified by the manufacturers to yield an output of 2.1 watts centered
at 253.7 nm. The tubes were mounted in a plane with their axes
parallel and 4 cm apart. To the rear of the tubes was a cylindrically concave reflecting head (Agar Products Ltd., Stansted, Essex, UK),
directing the UV light forward through a 6 × 24-cm rectangular aperture. The collagen solution was held in a quartz cuvette, placed
centrally in the beam with its front face positioned 1 cm from the
tangential plane connecting the front surfaces of the fluorescent tubes.
DSC Analysis
After measured irradiation times, 0.4 ml of solution was taken
from the cuvette and diluted to 0.4 mg/ml with 0.5 M acetic acid. The solution was stirred and degassed for 8 min using a Thermovac
apparatus (Microcal Inc., Northampton, MA) and scanned in a VP-DSC
(Microcal) from 10 to 60 °C. Numerical analysis of the data was
undertaken with the Microcal software using a cubic interpolation for
the base line and a "non-two-state" fitting procedure with cursor
initiation. To obtain convergence, it was necessary to provide initial
estimates of  Hv on the order of 105
cal/mol. Least squares fitting of the nonlinear function (Equation 2)
was performed using Origin Software.
Hydrogen Peroxide
To investigate the effect of increasing OH· radical
concentration during UV irradiation, approximately 0.3% hydrogen
peroxide (Sigma) was added to the collagen solution prior to irradiation.
Thiourea and Cysteamine
To examine the effect of reducing the free radical concentration
during UV irradiation, the free radical scavengers, thiourea and
cysteamine (Sigma), were added at concentrations of 1, 10, and 100 mM
SLS Crystal Formation
The irradiated solutions were examined to see whether the
collagen molecules were still capable of producing SLS crystals and
fibers. SLS crystals were prepared as follows. Solutions were diluted
to 0.2 mg/ml collagen in 0.05 M acetic acid and dialyzed against 0.4 g of ATP dissolved in 0.05 M acetic acid.
The crystallites were examined by transmission electron microscopy
(Philips 400). Other subsamples of the crystallites were degassed and
run in the VP-DSC, and the collagen content of further samples were
measured by hydroxyproline analysis both in the suspensions themselves, the pellet after centrifugation of a specified volume, and the supernatant. The latter tests were done to determine how much of the
sample produced the SLS crystals.
Chemical Analyses
Amino Acid Composition--
The composition of the
irradiated collagen was determined to investigate any specificity in
the degradation of the amino acids. The samples were hydrolyzed in 6 M hydrochloric acid and analyzed on an Alpha Plus II
Autoanalyzer (Amersham Pharmacia Biotech) using the standard program,
and detection of the amino acids was achieved by postcolumn
derivatization using ninhydrin.
Intermolecular Cross-links--
The presence of cross-links was
determined on the Alpha Plus using a modified gradient as
described previously in detail (16).
Hydroxyproline Assay--
The collagen content was determined by
the standard colorimetric assay (17) but employing the continuous
system from Chemlab based on the method of Grant (18).
Chemical Synthesis of Dityrosine
Dityrosine was synthesized by peroxide oxidation of tyrosine
according to the method described by Nomura et al.
(19).
End Group Analysis
Determination of new amino acid end groups exposed on cleavage
of the peptide chains was made by fluorescamine (Sigma). 0.2% (w/v)
fluorescamine in acetone was added to 0.4 M lithium
borate buffer, and fluorescence was read after 1 min using excitation at 390 nm and measuring emission at 475 nm, as described in detail (20). Preliminary measurements with known quantities of glycine (Sigma)
were used to calibrate the fluorescence reading in terms of numbers of
amide groups.
Polyacrylamide Gel Electrophoresis
The molecular weight changes were demonstrated by
SDS-polyacrylamide gel electrophoresis (21) followed by staining with Coomassie Blue; the stained gels were scanned using an Agfa Studioscan I flat bed scanner and Adobe Photoshop software; and the image was
analyzed using the NIH Image package.
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RESULTS |
Denaturation by a Rate Process--
When freshly prepared collagen
solutions were examined in the VP-DSC, the well known single sharp
helix-coil transition was seen at a temperature that varied linearly
with the logarithm of the scanning rate, as expected for a rate
process. The width at half-peak height increased linearly with the
scanning rate, consistent with the shape of the endotherm being
broadened slightly by the response time of the instrument.
Formation of an Intermediate State--
When samples were
irradiated and aliquots were examined at different irradiation times, a
new endotherm at approximately 32 °C appeared in addition to the
known peak for collagen at 39 °C (Fig.
1). The new peak grew with time of
irradiation while the first peak fell, and at about 1 h the former
was the only endotherm present. Further irradiation reduced this peak
progressively, and by about 2 h its area was less than 10% of its
maximum value. The two peaks, although easily resolvable, overlapped,
and to estimate the enthalpy of the two peaks separately it was
necessary to use a least squares fitting procedure (Microcal Inc.).
This showed a linear relation between the areas of the new and old peaks (Fig. 2), demonstrating that the
new peak was being created at the expense of the original triple
helical peak. The new peak appeared to be an intermediate state in the
degradation of collagen to random coils by UV following Scheme 1.
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Fig. 1.
UV-C degradation of collagen at 0 °C.
Solutions irradiated at 2 mg/ml and diluted to 0.4 mg/ml prior to
scanning. Numbers represent duration of exposure in
minutes.
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Fig. 2.
Linear relationship between the areas of the
intermediate state endotherms and native triple helix endotherms.
Circles, collagen solution in 0.5 M acetic acid
at 0 °C; squares, collagen solution in 0.5 M
acetic acid at room temperature; triangles, collagen
solution in 0.5 M acetic acid and 0.3% hydrogen peroxide
at room temperature.
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Since the least squares regression line cut the new peak axis at
approximately 80% of the enthalpy of the old peak (Table I), we can say that at least 80% of the
collagen followed this general scheme. Equally, the enthalpy of the
helix coil transition of the intermediate state must be at least 56 J/g, 80% that of the collagen triple helix.
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Table I
Linear relations between endotherm energies of the intermediate state
(y) and native triple helical state (x) during the initial period of
UV-C irradiation when the intermediate state endotherm increases at the
expense of that of the native state
Data of three experiments under different conditions are shown.
Experiments were as follows: A, collagen solution (2 mg/ml) in 0.5 M acetic acid at 0 °C; B, collagen solution (2 mg/ml) in
0.5 M acetic acid at room temperature; C, collagen solution
(2 mg/ml) in 0.5 M acetic acid and 0.3% hydrogen peroxide
at room temperature.
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The solution of the simultaneous differential equations governing the
above consecutive reactions is given for first order rate processes in
textbooks of physical chemistry (22), from which the concentrations
x, of triple helix, y, of the intermediate state,
and z, of the random coil state, can be written as
follows,
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(Eq. 1)
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(Eq. 2)
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(Eq. 3)
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where x0 is the initial concentration of
collagen. Eliminating t from Equations 1 and 2 yields the
following relation between x and y.
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(Eq. 4)
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Note that a Taylor expansion of this function at x = x0, yields a power series,
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(Eq. 5)
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that is approximately linear for small (x  x0)/x0, especially when
k2/k1 is small as in this work.
The concentrations were determined calorimetrically using measurements
of the areas under the DSC peaks, denoted as follows: QIS for the intermediate state,
QTH for the triple helical state, and
QTH0 for the triple
helical state of the unirradiated sample.
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(Eq. 6)
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Examples of these calorimetric measurements are plotted in Fig.
3. The lines represent least squares
fittings of the data to Equations 1-3. Numerical values for the fitted
rate constants k1 and k2
are given under various conditions in Table
II.
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Fig. 3.
Normalized concentrations of the native,
intermediate, and random coil states. Collagen solution is shown
at 0 °C.
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Table II
The rate constants k1 and k2 (s 1) estimated
in different ways and under different conditions
Experiments were as follows: A, collagen solution (2 mg/ml) in 0.5 M acetic acid at 0 °C; B, collagen solution (2 mg/ml) in
0.5 M acetic acid at room temperature; C, collagen solution
(2 mg/ml) in 0.5 M acetic acid and 0.3% hydrogen peroxide
at room temperature.
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Several lines of evidence confirmed that the majority of the native
molecules were denatured via the intermediate state. First, estimates
of the concentrations of the individual components followed closely the
time courses predicted by Equations 1-3 (see Fig. 3). Second, analysis
showed that the rate constant (k1) for the
degradation of the triple helix determined directly from Equation 1
agreed closely with the rate constant for the formation of the
intermediate state, determined independently (from Equation 2; see
Table II). Third, the ratio
k1/k2 determined either
directly using Equation 4 or by individual determinations of
k1 and k2 using Equation 2 agreed within the
experimental uncertainty (Table II). Finally, during the initial period
of irradiation, when intermediate state degradation was relatively
slight, the intermediate state endotherm grew as the native state
declined in approximately a 1:1 relationship, as predicted by the
Taylor approximation, Equation 5 (see Fig. 2). When the exact
expression was used to fit all of the data pooled, the enthalpy of
denaturation of the intermediate state was estimated as 70 ± 5 J/g, the same as the native state.
The temperatures declined and the widths of the endotherms at half
height increased with irradiation time under all conditions, and
partial denaturation by scanning to approximately
Tmax followed by rapid cooling showed an
elevated temperature and narrower width on rescanning (see Fig.
4). This demonstrated that each endotherm contained a distribution of molecules with different stabilities and
that this diversity increased with increasing doses of radiation. The
temperatures of both peaks were increased by increasing the scanning
rate or by reducing the pH.
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Fig. 4.
Evidence that the intermediate state is
heterogeneous. DSC thermograms of collagen solution (diluted to
0.4 mg/ml) in 0.5 M acetic acid after irradiation for
1 h at a collagen concentration of 2 mg/ml and at a temperature of
0 °C are shown. All scans were at 60 °C/h. Peak maxima are
indicated in degrees centigrade. Sample was scanned from 10 to 60 °C
and immediately cooled to 10 °C (a) and then rescanned
(b). A new sample was scanned from 10 to 30 °C and
immediately cooled to 10 °C (c) and then rescanned from
10 to 60 °C and immediately cooled (d) and rescanned
(e).
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Gelation--
During the initial period of irradiation (0-1 h)
the solution gradually gelled, indicating cross-linking. By 1 h, a
weak gel had formed, and the solution could no longer be poured from
the cuvette and could only be removed with a spatula. Vigorous stirring broke the gel. Prolonged irradiation reduced the strength of the gel,
and by 8 h, the solution was visibly of reduced viscosity.
Molecular Weight Change by Gel Electrophoresis--
Gel
electrophoresis showed a gradual reduction in the  -band,  -band,
and higher bands with UV dose (Fig. 5).
After 1 h of irradiation, the individual - or -bands were
barely discernible although the SDS and heat treatment had dissolved
all of the collagen. Samples exposed to UV for 1 h or longer
showed a continuum of dye along the whole length of the gel with a
concentration at the high molecular weight end (Fig. 5a).
Careful examination at low UV exposures (5 min) revealed some new
bands, below the -bands, indicating that chain scission may not be
entirely random (high density gels, not shown).
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Fig. 5.
SDS gel electrophoresis of collagen solutions
exposed to UV-C for different lengths of time. a,
lanes from left to right: 0 min, 1 min, 2 min, 5 min, 15 min, 30 min, 1 h. b, densitometry
of gel bands showing a decrease in intact 1 chains
(circles) and initial increase and subsequent decline in the
high molecular weight component (squares).
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Examination for higher molecular weight components, consistent with
gelling of the solution, revealed an initial increase in high molecular
weight components at the top of the gel, indicating extensively
cross-linked -chains, followed by a subsequent decline. It was
concluded that two processes were taking place simultaneously. The
chains had cross-linked but had also been cut predominantly randomly
along their length so that the resulting population of chain lengths
was distributed down the length of the gel.
Chain Cleavage Determined by Fluorescamine End Group
Analysis--
The fluorescamine analysis showed that amide groups
increased at a rate of 3.7 ± 0.96 mol of amide/mol of -chain
during the first 3 h of UV exposure, confirming that chain
cleavage was occurring. This rate constant was of the same order as the
rate constant for intermediate state production (Table II) and the rate
at which the -chains visibly disappeared from the gels (Fig. 5).
Formation of SLS Crystallites--
The experiments to determine
whether the intermediate state of the irradiated collagen was
sufficiently intact to be capable of forming SLS crystals revealed the
presence of SLS crystals in the electron microscope (Table
III). Measurements of the electron micrographs showed that these crystals were of the same length as
crystals formed from unirradiated collagen (Table III) but possessed a
less ordered internal architecture (Fig.
6). The DSC study showed that the
crystals of irradiated collagen were less stable thermally than the
controls (Table III).
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Table III
Properties of the SLS crystal suspensions prepared from collagen
solutions irradiated for different lengths of time
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Fig. 6.
Electron micrographs of SLS crystals prepared
from collagen solutions before and after 1-h UV-C irradiation.
a, unirradiated solution (i.e. native state).
b, solution irradiated for 1 h (i.e.
intermediate state and random chains). Bars, 64 nm.
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Acceleration and Inhibition of Hydroxyl Radicals--
The addition
of hydrogen peroxide to the solution increased both
k1 and k2 by over
10-fold. This showed that increasing the concentration of OH·
radicals, which are produced by the splitting of the
H2O2 molecule when illuminated by ultraviolet
light (23), increased both the rate of formation of the intermediate
state and its degradation into random coils (see Fig.
7, Table II). Equally, the addition of
the free radical scavengers, cysteamine or thiourea, reduced both
k1 and k2. Thiourea was
particularly effective and at a concentration of 100 mM
suppressed all intermediate state production under the conditions of
these experiments (Fig. 8). There was an
approximately linear relation between log k1 and
log k2 (Fig.
9).
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Fig. 7.
UV-C degradation of collagen solutions (2 mg/ml) in the presence of 0.3% hydrogen peroxide.
Numbers represent duration of exposure in minutes. All
solutions diluted to a collagen concentration of 0.4 mg/ml prior to
scanning.
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Fig. 8.
UV-C degradation of collagen in the presence
of OH· radical scavengers or an OH· radical
generator. Data for 5-min exposure to UV-C of collagen solution (2 mg/ml) in 0.5 M acetic acid with different additives as
follows: 100 mM thiourea (a); 100 mM
cysteamine (b); no additions (c); 0.3%
H2O2 (d). All solutions were diluted
to collagen concentrations of 0.4 mg/ml prior to scanning.
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Fig. 9.
Relation between the rate constants for
intermediate state formation (k1) and
degradation (k2). Each point represents
measurements of collagen solution (2 mg/ml) in 0.5 M acetic
acid with different additives as follows.  , 1 mM
cysteamine;  , 10 mM cysteamine;  , 100 mM
cysteamine;  , no additives;  , 0.3% H2O2;
+, 100 mM cysteamine plus 0.3%
H2O2; ×, 100 mM thiourea plus
0.3% H2O2. All experiments were conducted at
room temperature. The line represents the least squares
regression line for all of the data pooled: log10
k2 = (1.136 ± 0.087)log10
k1 0.288 ± 0.262.
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Chemical Composition--
Amino acid analysis revealed a rapid
loss of tyrosine (50% in 1 h) and an even more rapid loss of
phenylalanine (70% in 1 h), while other amino acids were
virtually unaffected.
Formation of Unknowns--
We could not detect dityrosine, using
an authentic sample synthesized in the laboratory, or DOPA (Sigma),
both of which have previously been reported to be present in irradiated
collagen (2, 3). Two unknown peaks were present in the "cross-link region" of the chromatogram between tyrosine and hydroxylysine, and
both increased with time of irradiation (Fig.
10). Attempts are currently being made
to isolate and characterize these two components.
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Fig. 10.
Elution profiles from the amino acid
analyzer using the modified gradient showing the components in the
"cross-link region." The increase in the unknown group with
irradiation time is compared with the location of dityrosine and DOPA.
a, nonirradiated collagen; b, collagen irradiated
for 8 h showing the presence of unknown components. c
and d, locations of authentic dityrosine and DOPA,
respectively.
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DISCUSSION |
We have shown that ultraviolet light reduces the native triple
helical collagen to random chains via an intermediate state of slightly
lower thermal stability, at a Tm of around 32 instead of 39 °C.
The high enthalpy of denaturation and the highly cooperative thermal
unfolding of the intermediate state are characteristic features of the
triple helix and are therefore consistent with the intermediate state
being mainly triple helical. Since the gels showed loss of both -
and -bands together, we interpret the loss as being caused by chain
scission. The gel analysis (Fig. 5) revealed that no intact -chains
remained after 1 h of irradiation, when about 60% of the collagen
was in the intermediate state (Fig. 3). Chain scission is known to
destabilize collagen (24). Thus, we suggest that the intermediate state
is a triple helix destabilized, at least partly, by UV-induced chain
scission. While the triple helix remains intact, due to hydrogen
bonding, a small number of chain scissions would be expected to cause
little change in the enthalpy of denaturation, since most of the
interchain hydrogen bonds would remain unaffected. The reduction in
denaturation temperature would be caused mainly by entropic effects.
Irradiated preparations, containing only intermediate state with very
little native triple helix, still produced high yields of SLS
crystallites, confirming that the intermediate state was triple helical
and that the damaged triple helix was the same length as the native
molecule (Table III). The intermediate state SLS crystals were less
thermally stable than the native SLS crystals (Table III), reflecting
the fact that the molecules themselves were intrinsically less stable,
due to UV damage. The molecules in SLS crystals possessed a higher
denaturation temperature than the same molecules in solution due to
intermolecular interactions, as observed in fibers (14).
The formation of the intermediate state and the disappearance of the
1- and 2-chains on the gels (see Fig. 5)
occurred at similar rates (compare Fig. 5 and Table II). We suggest
that chain scission is predominantly random, occurring at many possible
sites along the length of the chains as, by about 30 min of
irradiation, the gels showed a rather uniform smearing out of the bands
down the gel. However, we note that there is some evidence initially of
specific scission with new bands occurring on the gels just below the
-bands. We suggest that these may be associated with parts of the
molecule that are relatively sensitive to OH· radical attack.
These active radicals are presumably produced from
H2O2 and
O2.
The substantial increase in k1 and
k2 that was observed upon introducing hydrogen
peroxide into the sample was presumably caused by increased
concentrations of OH· radicals induced by the splitting of the
hydrogen peroxide molecule into two OH· radicals by ultraviolet
light (23).
The OH· radicals clearly induce chain scission, which leads to
loss of thermal stability. The involvement of OH· radicals,
which would be present along the whole length of the collagen molecule,
would explain why the cutting of the -chains is predominantly random
at points along the chain. This explanation was reinforced by
observations of the effect of the OH· radical scavengers
thiourea and cysteamine, which substantially reduced
k1 and k2 and chain
scission (gels not shown) even in the presence of hydrogen peroxide.
These scavengers also reduced UV damage even when no peroxide was
added; thiourea was so effective that at a concentration 100 mM there was no resolvable loss in thermal stability and no
intermediate state even after 1-h irradiation (Fig. 8).
Each scission event will cause more or less loss of thermal stability
depending on its position along the chain. Our suggestion that the
degradation of collagen to random coils is via the action of free
radicals causing random scission along the length of all the -chains
is consistent with the observation that the endotherms at 39 and
32 °C are caused by a population of molecules that increase in
heterogeneity and fall in thermal stability as the irradiation proceeds
(Fig. 4). We suggest that the effect of several chain scissions reduces
Tm by opening up the helix. We speculate that the
intermediate state is formed once the accumulated damage destabilizes
the molecule beyond a critical level, which causes the transition
temperature to flip from ~39 to ~32 °C, i.e. to flip
to the intermediate state. Since the rate constant for generating the
intermediate state is faster than that for its subsequent degradation,
we deduce that the average number of scissions yielding the
intermediate state is less than the average number of further scissions
required to reduce the intermediate state to random coils.
By drawing all of the results together, we can begin to piece together
the possible sequence of events by which UV light reduces collagen
molecules to random coils via an intermediate state. The primary effect
of UV light is to generate free radicals in the water molecules
surrounding the collagen molecule, and these radicals react with the
collagen, destabilizing it. At least one of these reactions causes
chain scission, which can occur at many sites along the length of the
molecule, and the selection of these sites is predominantly random. The
number of intact -chains in the population of collagen molecules
therefore declines, as observed in the gels, and the number of scission
points increases, as observed. While there is evidence from the gels of
some new bands being formed, indicating that some sites along the
-chains are more likely to be cut than others, the cutting is
predominantly random. Thus, the majority of the cut -chain matter
was smeared out along the whole length of the gel. As irradiation
proceeds, the number of undamaged molecules falls and the number of
damaged molecules rises. A population of new molecules with different
damaged sites is produced, and these molecules have slightly different
thermal stabilities, broadening the denaturation endotherm and reducing Tmax. Provided the damage is less than a
certain critical level, the mechanism by which the triple helix unfolds
is basically unaltered, requiring the initial uncoupling of the
-chains of the major thermally labile unit at the C end of the
molecule (15), followed by the rapid unzipping of the three chains
along the length of the molecule. The enthalpies of activation are
therefore the same in these slightly damaged molecules, and the
reductions in stabilities are caused by an increase in the entropy of
activation resulting from the increased flexibility of the damaged
helix. Once sufficient damage has been inflicted, beyond the
hypothetical critical level, the molecule becomes so unstable that the
denaturation temperature flips, from its value around 39 °C to a new
temperature around 32 °C. This is because the unzipping of the
intermediate state needs fewer bonds to be broken initially to produce
the required free energy of activation. The precise amount of damage
that corresponds to the critical level has not been defined by these
experiments, but comparison of the rate at which the number of
undamaged chains declines in gels with the rate at which the
intermediate state forms indicates that the critical level corresponds
with at least two or all three chains being cut within the molecule.
Thus, the single scission of a single chain is not sufficient to yield
random coils, and the process of degradation of the native triple helix proceeds indirectly via an intermediate state. This intermediate state
is basically triple helical and the same length as the unirradiated molecule, but with cuts in the -chains. It therefore has an enthalpy of denaturation very close to that of native collagen, shows a highly
cooperative denaturation process, and produces SLS crystals of the same
length as those of native collagen.
Further scission of the chains in the intermediate state destabilizes
it even further. The Tmax of the
denaturation endotherm therefore falls, while its width increases due
to increasing heterogeneity in stability of the population of molecules
comprising the intermediate state. Some scissions are sufficient to
reduce part, or the whole, of the intermediate state molecule to random
coils. With increasing irradiation times, the pool of molecules with a
denaturation temperature around 39 °C declines to zero as they are
reduced to the intermediate state, and all that remains are random
chains and intermediate state. Finally, degradation of the intermediate
state through chain scission continues until all of the molecules are
reduced to random chains. The number of chain scissions required to
cause complete disruption of the triple helical structure we estimate to be about 3 per chain on average, based on comparison with the gels.
Since generation of the intermediate state requires on average about
one cut per chain, the rate constant k2 is
always smaller than k1.
In summary, we have shown that the collagen molecule can be quite
extensively damaged by cleavage of the primary peptide bonds without
disorganizing the triple helical structure. This leads to the formation
of a damaged "intermediate state" prior to degradation of the
molecules to random chains. The initial gelation of the solution
indicates that both cross-linking and chain cleavage are occurring but
that the prevalent reaction is chain cleavage by hydroxyl radicals. An
understanding of the mechanisms involved will be of considerable value
in future studies of the effects of UV on dermal collagen in photoaging.
| |
ACKNOWLEDGEMENT |
We thank Anne Phillips for undertaking
the electron microscopy.
| |
FOOTNOTES |
*
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.
§
To whom correspondence should be addressed. Tel.: 44 117 928 9245;
Fax: 44 117 928 9505; E-mail: chris.miles@bristol.ac.uk.
Published, JBC Papers in Press, July 11, 2000, DOI 10.1074/jbc.M002346200
| |
ABBREVIATIONS |
The abbreviations used are:
DOPA, 3,4-Dihydroxyphenylalanine;
DSC, differential scanning
calorimetry;
SLS, segment long spacing.
| |
REFERENCES |
| 1.
|
Fujimori, E.
(1988)
FEBS Lett.
235,
98-102
|
| 2.
|
Kato, Y.,
Uchida, K.,
and Kawakishi, S.
(1994)
Photochem. Photobiol.
59,
343-349
|
| 3.
|
Kato, Y.,
Takashi, N.,
and Shundro, K.
(1995)
Photochem. Photobiol.
61,
367-372
|
| 4.
|
Kaminska, A.,
and Sionkowska, A.
(1996)
Polymer Degradation Stability
51,
19-26
|
| 5.
|
Sionkowska, A.,
and Kaminska, A.
(1999)
Int. J. Biol. Macromol.
24,
337-340
|
| 6.
|
Bailey, A. J.
(1967)
Radiat. Res.
31,
206-214
|
| 7.
|
Hayashi, T.,
Curran-Patel, S.,
and Prockop, D. J.
(1979)
Biochemistry
18,
4182-4187
|
| 8.
|
Harrington, W. F.,
and von Hippel, P. H.
(1969)
Adv. Protein Chem.
16,
1-138
|
| 9.
|
Burjanadze, T. V.,
and Bezhitadze, M. O.
(1992)
Biopolymers
32,
951-956
|
| 10.
|
Danielsen, C. C.
(1990)
Biochem. J.
272,
697-701
|
| 11.
|
Rose, C.,
Kumar, M.,
and Mandal, A. B.
(1988)
Biochem. J.
249,
127-133
|
| 12.
|
Privalov, P. L.,
Tiktopulo, E. I.,
and Tischenko, V. M.
(1979)
J. Mol. Biol.
127,
203-217
|
| 13.
|
Tiktopulo, E. I.,
and Kajava, A. V.
(1998)
Biochemistry
37,
8147-8152
|
| 14.
|
Miles, C. A.,
and Ghelashvili, M.
(1999)
Biophys. J.
76,
3243-3252
|
| 15.
|
Miles, C. A.,
Burjanadze, T. V.,
and Bailey, A. J.
(1995)
J. Mol. Biol.
245,
437-446
|
| 16.
|
Sims, T. J.,
and Bailey, A. J.
(1992)
J. Chromotogr.
582,
49-55
|
| 17.
|
Bergman, I.,
and Loxley, R.
(1963)
Anal. Chem.
35,
1961-1965
|
| 18.
|
Grant, R. A.
(1964)
J. Clin. Pathol.
17,
685-686
|
| 19.
|
Nomura, U.,
Suzuka, N.,
and Matsumoto.
(1990)
Biochemistry
29,
4525-4534
|
| 20.
|
Smolenski, K. A.,
Avery, N. C.,
and Light, N. D.
(1983)
Biochem. J.
213,
525-532
|
| 21.
|
Laemmli, U.
(1970)
Nature
227,
680-685
|
| 22.
|
Atkins, P. W.
(1978)
Physical Chemistry
, pp. 868-869, Oxford University Press, Oxford
|
| 23.
|
Halliwell, B.,
and Gutteridge, J. M. C.
(1999)
Free Radicals in Biology and Medicine
, 3rd Ed.
, Oxford University Press, Oxford
|
| 24.
|
Rossi, A.,
Zanaboni, G.,
Cetta, G.,
and Tenni, R.
(1997)
J. Mol. Biol.
269,
488-493
|
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