J. Cell Sci. 7, 387-405 (1970)
Printed in Great Britain
387
THE EFFECTS OF IRRADIATION WITH HIGH
ENERGY ELECTRONS ON THE STRUCTURE
AND REACTIVITY OF NATIVE AND
CROSS-LINKED COLLAGEN FIBRES
R. A. GRANT, R. W. COX AND C. M. KENT
Agricultural Research Council, Institute of Animal Physiology, Babraham, Cambridge,
England
SUMMARY
Native and cross-linked rat tail tendon collagen was irradiated with high energy electrons up
to a maximum dose of 100 x io 4 J kg" 1 (100 Mrd). Fibres irradiated dry showed greater damage
when examined in the electron microscope using negative staining techniques than those
irradiated wet. Cross-linking with glutaraldehyde prior to irradiation resulted in the band
structure being preserved even at the highest dose but an unequal shrinkage of the bands was
noted.
Irradiation altered the reactivity of both native and cross-linked collagen with collagenase
and elastase. Wet- but not dry-irradiated native collagen became resistant to collagenase.
Both wet- and dry-irradiated specimens were digested with elastase. Cross-linked collagen,
normally resistant to both elastase and collagenase, became sensitive after varying doses of
radiation; different results were obtained after irradiation in the wet and dry states.
Irradiated collagen reacted abnormally with various histological stains and tended to resemble
elastin in tinctorial properties.
No marked changes were noted in the amino acid composition of the collagen after irradiation.
Solubility in dilute alkali, acetic acid and hot water was decreased after irradiation of collagen
in the wet state and increased after irradiation in the dry state.
The results were consistent with the hypothesis that electron irradiation of collagen in the dry
state results in scission of the polypeptide chains and that, in the presence of water, this is
accompanied by the formation of intermolecular bonds. It also appears that changes in the
configuration of the polypeptide chains accompany both these processes.
INTRODUCTION
In recent years a number of investigations have been made on the effects of ionizing
radiation on various proteins, including collagen.
Although collagen is distributed throughout the body, it is usually present in small
and variable amounts in most tissues. Consequently, studies on the effects of irradiation on collagen have usually been made on tendon where it occurs in a relatively
pure state.
Bailey, Bendall & Rhodes (1962) studied the effect of irradiation with 2 MeV
electrons on the shrinkage temperature of rat tail tendon collagen. Doses up to
4oxio 4 jkg~ 1 (40 Mrd) were given at o °C. It was found that the hydrothermal
shrinkage temperature decreased progressively with dose while the amount by which
the fibres shortened under a fixed load increased from 6% for native collagen to a
25
C E L7
388
R. A. Grant, R. W. Cox and C. M. Kent
maximum of 70% for collagen treated with doses greater than 6 x io 4 J kg"1. Irradiation under various conditions led to the conclusion that these effects were due to the
direct action of the radiation on the protein molecule and not to subsequent chemical
attack by radicals from water.
Bailey & Tromans (1964) investigated the effects of electron radiation on the ultrastructure of collagen fibrils using the electron microscope and a negative staining
technique. Below i o x i o 4 j k g - 1 , no irradiation-induced effects were observed but
higher doses resulted in a gradual loss of the band contrast of the fibrils, and at
40 x io4 J kg"1 the cross-banding had completely disappeared. The effects of irradiation on the fibrils were compared with those of other treatments such as the action of
urea and various cross-linking chemicals. It was concluded that loss of band pattern
was primarily due to swelling of the fibrils resulting from a structural disorganization
of the collagen macromolecule and the subsequent uptake of water. Bailey, Rhodes &
Cater (1964) found that intermolecular cross-links were produced when rat tail
tendon collagen was irradiated in the presence of water, whereas, in contrast, irradiation in the dry state resulted in fragmentation of the macromolecules; no histological
or enzymic observations were made on the irradiated collagen.
A detailed examination of the effects of electron radiation on the tensile strength of
bovine tendon was made by Braams (1961, 1963). He found that for dry tendon the
dose required to reduce the tensile strength to 37% of its original value was approximately i 8 x i o 4 j kg"1. For hydrated tendon, reduction of tensile strength by the
same amount required 46 x io 4 J kg"1.
Other less detailed studies have been made of the effects of electron irradiation on
collagen. Perron & Wright (1950) found that changes in the physical appearance of
kangaroo tail tendon were less marked in tendon irradiated dry rather than wet.
However, when the dry-irradiated material was subsequently immersed in water
most of the material dissolved, leaving only a thin gelatinous film that gave an amorphous low angle X-ray diffraction pattern.
Kuntz & White (1961) reported that cross-linking predominated over chain scission
during irradiation with electrons of collagen in the wet state.
The object of the present communication was to extend previous studies of the
effects of electron irradiation on collagen by (1) using a wider dose range, (2) including
cross-linked in addition to native collagen and (3) investigating changes in the histological staining reactions of the irradiated collagen and its reactivity with collagenase
and elastase.
MATERIALS AND METHODS
Collagen preparations
Freshly dissected rat tail tendons were washed in cold saline (0-9 % NaCl) and stored in cold
saline until required. Hydroxyproline analysis showed that the fibres consisted of almost pure
collagen. Fibres to be irradiated in the dry state were dried under vacuum in a desiccator
before being irradiated in narrow, stoppered glass tubes; the material to be irradiated wet was
placed in tubes containing saline. The sample tubes were suspended in a beaker containing ice
and water, and during irradiation at the higher dose levels the ice was replaced at intervals.
Electron irradiation of collagen
389
Cross-linked collagen was prepared by immersing the fibres in cold neutral 5 % glutaraldehyde solution overnight; the fibres were then carefully washed and part of the preparation dried
in vacuo.
Irradiation
The beaker containing the specimen tubes was placed directly in the beam from a 14 MeV
linear accelerator, the dose level being varied over the range 2-100 x io4 J kg"1 (2-100 Mrd).
The temperature rise inside the specimen tubes was measured using a thermocouple. Even at
the highest doses the temperature of the samples immersed in saline did not rise above 20 °C,
which is well below the denaturation temperature of collagen; the temperature of the dry specimens could not be measured in this way, but there was no evidence that these reached a higher
temperature than the wet samples.
Enzyme reactions
Portions of the irradiated collagen were incubated at 37 °C with crystalline elastase solution
(1 mg/ml) (Lewis, Williams & Brink, 1956) adjusted to pH 9-0 with tris buffer. Samples were also
incubated at pH 7-0 with bacterial collagenase (3 mg/ml) (Worthington Biochemical Corp.)
from which the elastase activity had been removed by adsorption on powdered elastin overnight at 4 °C followed by removal of elastin by centrifuging. The degree of digestion was
estimated by visual inspection.
Solubility
Samples of irradiated and control rat tail tendon were placed in 0-5 % acetic acid and allowed
to stand overnight at room temperature. Solubility in hot water was tested by heating samples of
tendon fibres with water in an autoclave at 103-4 kN m~2 (15 lb in.~2) pressure for 6 h. Further
samples were heated at 95 °C in O'i N NaOH solution until dissolved and the times required
for complete solution recorded.
Histology
Specimens of irradiated and control rat tail tendon werefixedin 1 o % formol saline, embedded
in paraffin and sectioned. Sections were stained with orcein, Van Gieson stain, phosphotungstic
acid/haematoxylin, Luxol fast blue and Weigert's elastic stain.
Amino acid analysis
Accurately weighed dry samples of collagen were hydrolysed by heating with 6 N HC1 for 6 h
in sealed tubes in an autoclave at 1034 kN m~2 (15 lb in.~2) pressure. The resulting hydrolysates were then analysed by ion exchange chromatography using a Technicon amino acid
analyser. Only the specimens treated with 100 x io4 J kg~L irradiation (wet and dry, native and
cross-linked) and the corresponding untreated controls were analysed for amino acids.
Electron microscopy
Small portions of tendon were ground in 1 % ammonium acetate solution to give a fine
suspension. The finely dispersed material was negatively stained on grids with a solution of
2 % phosphotungstic acid that had been adjusted to pH 7-4 by the addition of KOH (Brenner &
Home, 1959). The preparations were examined in a Siemens Elmiskop I electron microscope at
an accelerating voltage of 80 kV and at machine magnifications of 40000, 47000 and 80000.
The microscope had been calibrated using negatively stained beef liver catalase crystals as a
standard, allowances being made for the variables causing errors in magnification (Elbers &
Pieters, 1964; Cox & Home, 1968).
25-2
R. A. Grant, R. W. Cox and C. M. Kent
39°
RESULTS
Enzyme reactions
Native wet fibres (Figs, i, 2). Irradiation of collagen fibres in the wet state with
doses up to 25 x io4 J kg"1 did not affect sensitivity to bacterial collagenase, complete
digestion of the material being obtained. At 50 x io 4 J kg"1 no digestion was noted
after 24 h but after 5 days the sample was completely digested. With a dose of
100 x io4 J kg"1 no digestion was obtained with collagenase even after 5 days. At a dose
level of 2 x io4 J kg"1 the treated collagen was not attacked by elastase but at dose
levels above 25 x io 4 J kg"1 it was completely digested by this enzyme.
•S
100 -
100 -
75
75
.9
50
50
W
to
CJ
Q
25
0 -
0 25
50
100
Dose of electrons (x10 4 J kg"1)
(1 Mrd = 104J kg"1)
Fig. 1
Fig.
O Fig.
O -
25
50
100
Dose of electrons (x 104 J kg"1)
(1 Mrd = 104J kg"1)
Fig. 2
1. Effect of collagenase on irradiated native collagen at pH 7 0 , 37 °C for 24 h.
- O, dry; •
• , wet.
2. Effect of collagenase on irradiated native collagen at pH 7 0 , 37 °C for 5 days.
- O, dry; •
• , wet.
Nativefibresirradiated in the dry state (Figs. 1, 2). Irradiation with electrons even at
the highest dose of 100 x io4 J kg"1 of fibres in the dry state did not result in any loss
of sensitivity of the collagen to bacterial collagenase, in contrast to the results obtained
in the wet state. However, all the treated samples were completely digested by elastase
except for those which received the lowest dose of 2 x io 4 J kg"1, which were resistant.
Thus, irradiation with electrons in the dry as well as in the hydrated state results in
collagen becoming sensitive to elastase to which it is normally resistant.
Cross-linkedfibresirradiated in the wet state (Figs. 3-6). Collagen cross-linked with
glutaraldehyde is usually completely resistant to the action of bacterial collagenase.
Irradiation with electrons in the wet state resulted in the material becoming sensitive
to the action of collagenase although digestion was very slow. No effect was noted
Electron irradiation of collagen
391
4
1
after 24 h but after 5 days the samples which had received 50 x io J kg" of
radiation were partially digested by the enzyme while the samples which had received
25 and 100 x io4 J kg"1 were not significantly attacked.
Fibres which received a 2 x io4 J kg"1 dose in the wet state were not digested with
elastase. Those treated with 25 and 50 x io 4 J kg"1 were partly digested after 24 h and
completely digested after 5 days while the fibres receiving the highest dose were not
attacked even after 5 days.
100
100 -
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Dose of electrons (x 104 J kg"')
(1 Mrd = 104J kg"')
0 100
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Dose of electrons (xiO* J kg"')
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Fig. 3
Fig- 4
Fig. 3. Effect of collagenase on irradiated cross-linked collagen at pH 7-0, 37 °C for
24 h. O - - O, dry; •
• , wet.
Fig. 4. Effect of collagenase on irradiated cross-linked collagen at pH 70, 37 °C for
5 days. O - - O» dry; #
# , wet.
Cross-linkedfibresirradiated in the dry state {Figs. 3-6). Irradiation of cross-linked
collagen fibres in the dry state at high dose levels resulted in their becoming sensitive
to the action of collagenase, although digestion was slow. No effect was noted after
24 h incubation at any dose level. However, after 5 days incubation the collagen
treated with 5oxio 4 Jkg~ J was partially digested and the specimen treated with
100 x io4 J kg"1 was completely digested.
Elastase had no action on cross-linked collagen irradiated at the 2 x io4 J kg"1 level
in the dry state. At the 25Xio 4 Jkg~ 1 level no digestion was obtained even after
5 days incubation while, with 50 x io 4 J kg"1, the sample was partially digested by
elastase after 24 h and completely dissolved after 5 days. The specimen that received
the i o o x i o 4 j k g " 1 dose was completely digested after 24 h. These results are in
almost complete contrast to those found with the cross-linked fibres irradiated in the
wet state.
Effect of autoclaving on irradiated cross-linked collagen (Figs. 7, 8). Whereas, in both
R. A. Grant, R. W. Cox and C. M. Kent
392
100 h
100
75
s
75
50
(U
Q
25
-
i
100
25
50
Dose of electrons (x10 4 J kg"1)
(1 Mrd = 104 J kg"1)
25
50
100
Dose of electrons (x 10" J kg"')
1
(1 Mrd = 10" J kg" )
Fig. 6
Fig. S
Fig. 5. Effect of elastase on irradiated cross-linked collagen at pH 9-0, 37 °C for 24 h.
A - - A, dry; A
A, wet.
Fig. 6. Effect of elastase on irradiated cross-linked collagen at pH 9-0, 37 °C for
5 days. A - - A, dry; A
A, wet.
D
25
50
100
Dose of electrons (x10"J kg"1)
(1 Mrd=10 4 J kg"1)
Fig. 7
25
50
Dose of electrons (x 104 J kg"1)
(1 Mrd=10 4 J kg"1)
Fig. 8
Fig. 7. Effect of elastase on autoclaved irradiated cross-linked collagen at pH 9-0,
37 °C for 24 h. A - - A, dry; A——A, wet.
Fig. 8. Effect of collagenase on autoclaved irradiated cross-linked collagen at pH 7-0,
37 °C for 24 h. O - - O, dry; « - — « , wet.
100
Electron irradiation of collagen
4
393
1
the wet and dry state, samples which had received 25 x io J kg" were resistant to the
action of collagenase, they were completely digested within 24 h after autoclaving with
water under the conditions specified. At a level of 5oxio 4 jkg" 1 , autoclaving increased the rate of digestion by collagenase. The degree of digestion after 24 h was
about the same for both wet- and dry-irradiated samples as was obtained after 5 days
where the specimen had not been autoclaved. However, autoclaving had no effect on
the specimens given 100 x io4 J kg"1 in the wet state, these being undigested even
after 5 days.
Autoclaving with water markedly increased the rate of digestion by elastase of wetirradiated, cross-linked collagen at the 25xio 4 Jkg" ] level but had no apparent
effect on the same material treated with 100 x io 4 J kg"1 which appeared unaffected.
In the case of cross-linked collagen irradiated dry with 25 x io 4 J kg"1, this was not
attacked by elastase but, after autoclaving, was completely digested. With a dose of
50 x io 4 J kg"1 the rate of digestion was markedly increased following autoclaving,
while at the 100 x io4 J kg"1 level autoclaving had no apparent effect on the reactivity
with elastase, complete digestion being obtained in 24 h.
Histological staining reactions
In general, irradiation with electrons resulted in the collagen fibres staining abnormally. This was so even though the temperature was maintained at a low level (below
20 °C) during irradiation. With irradiated doses above 25xio 4 Jkg~ 1 the treated
collagen tended to resemble elastin in its tinctorial reactivity. This result was obtained
with fibres irradiated in the dry as well as in the hydrated state. Fibres irradiated with
doses above 25 x io4 J kg"1 stained yellow rather than red with the Van Gieson stain,
and the affinity for orcein was increased, although at no dose level did the treated
collagen stain as intensely as elastin. Very marked effects were noted in the case of the
phosphotungstic acid/haematoxylin stain, the treated fibres usually tending to assume
a deep purple colour in contrast to the red staining of the control material. With
haematoxylin and eosin the irradiated fibres were usually stained mauve, in contrast to
the light pink of the controls, and with Weigert's elastic stain the treated fibres tended
to resemble elastin rather than collagen. Irradiated fibres also stained abnormally
with Luxol fast blue, often appearing pale grey or mauve instead of the clear, deep
blue of the controls.
Solubility
All the irradiated specimens of native collagen were soluble when autoclaved with
water at 103-4 kNm~2 (15 lb in."2) pressure except the specimen which received
iooxio 4 jkg~ 1 in the wet state. The irradiated cross-linked specimens were all
insoluble when autoclaved with water.
Solubilities in dilute sodium hydroxide solution and acetic acid are shown in Tables 1
and 2.
394
R- A. Grant, R. W. Cox and C. M. Kent
Amino acids
No marked differences were found in the amino acid compositions of irradiated and
control samples with both native and cross-linked collagen. Collagen cross-linked
with glutaraldehyde showed an 80% drop in the lysine and hydroxylysine contents.
Table 1. Solubility of native and cross-linked collagen, wet- and dry-irradiated
and non-irradiatedfibresin o-i N NaOH at 95 °C
Treatment
Native fibres
Cross-linked
fibres
25
X
IO 4 J kg" 1 50 x io 4 J kg" 1 100 X
(Wet
11*>
11
I Dry
2
2
300
/Wet
300
240
\Dry
195
#> time in min for complete solution.
IO4
J kg" 1
Control
60
17
2
260
210
310
—
Table 2. Solubility of native and cross-linked collagen, wet- and dry-irradiated
and non-irradiatedfibresin 0-5% acetic acid at room temperature
25 x io 4 J kg" 1
Treatment
Wet
SJative fibres
Dry
Cross-linked
IWet
fibres
1 Dry
Slightly
swollen
50 x io 4 J kg" 1 100 x io 4 J kg" 1
N
N
C
C
C
25*
10
5
N
N
N
N
N
N
Control
120
N
C, complete solubility; N, insoluble; *, time in min for complete solution.
Electron microscopy
Native collagen. There was progressive loss of contrast in the bands with increasing
dose of radiation. At a dosage level of 25 x io 4 J kg"1, collagen that had been irradiated
in the dry state was markedly affected (compare Figs. 9 and 11). Most of the fibrils
had lost their cross-striations. Careful search, however, revealed occasional traces of
cross-striation. Collagen that had been irradiated in the wet state with 25 x io4 J kg"1
was not affected to the same degree. The changes ranged from small, irregular,
localized areas of swelling with poor definition of the cross-striations to large lengths
of swollen fibrils with no trace of cross-striations. Nevertheless, in all samples,
evidence of cross-striation could be found somewhere along the length of a fibril.
With dosages of 50 x io 4 J kg-1, cross-striations had completely disappeared from
collagen that had been irradiated in the dry state. Much of the collagen did not even
have a fibrous appearance; it was present as amorphous, irregular masses. Collagen
that had been irradiated at the same dosage level but in the wet state showed less
alteration (Fig. 13). The changes varied from localized, swollen areas with loss of crossstriations, through generally swollen fibrils with perceptible but poorly defined crossstriations, to definable fibrils devoid of any cross-striations.
Electron irradiation of collagen
395
With dosages of 100 x io4 J kg"1 collagen that had been irradiated in the dry state
showed neither formed fibrils nor any suggestion of cross-striations. The material
was amorphous and irregular (Fig. 12). In collagen irradiated with the same dosage in
the wet state the changes were less severe (Fig. 14). Although amorphous and
irregular masses of material were present the shape of the fibrils was mainly
preserved. Many of these fibrils possessed no cross-striations and were irregularly
swollen. In others traces of cross-striations remained.
Cross-linked collagen. The protection afforded by cross-linking with glutaraldehyde
against the effects of irradiation was considerable. With dosages below 30 x io 4 J kg"1
there was no change in the morphology of the collagen irradiated in either the wet or
the dry states (compare Figs. 10 and 15). Protection appeared to be complete.
At a dosage level of 50 x io4 J kg"1 appreciable changes were present. The collagen
that had been irradiated in the wet state showed some localized swelling of the fibrils
with relatively poor definition of the cross-striations in those areas (Fig. 16). The
remainder of a fibril might be unaffected. The collagen irradiated in the dry state
with the same dosage showed a more generalized poor definition of the cross-striations
(Fig. 18) and also a shortening of the repeat period of the fibrils.
With dosages of 100 x io4 J kg"1 the collagen irradiated in the wet state exhibited a
more widespread decrease in the definition of the cross-striations than at 50 x io 4 J kg"1
in the wet state together with a shortening of the normal repeat period (Fig. 17).
Collagen irradiated in the dry state with i o o x i o 4 j k g " 1 showed more marked
changes than collagen irradiated wet at the same dose levels. Swelling and loss of
definition of cross-striations were more widespread. Nevertheless, occasional fibrils
were remarkably well preserved (Fig. 19).
Although the differences between cross-linked collagen irradiated in the wet and
dry states were not as marked as those between native collagen irradiated wet and dry,
the cross-linked collagen irradiated in the dry state continued to be more affected
morphologically than that irradiated in the presence of water at the same dosage.
To illustrate the difference between native and cross-linked collagen, it may be
noted that the changes in native collagen that had been irradiated in the wet state at
dosages of 32 x io4 J kg"1 were always greater than those in cross-linked native collagen
that had been irradiated in the wet state at dosages of 100 x io4 J kg"1.
DISCUSSION
Structural changes
Negative-staining techniques reveal the tropocollagen macromolecules as thin
filaments of approximately 1-5 nm diameter running roughly parallel to the direction
of the intact fibril. The alternating sequence of light (A) and dark (B) bands along the
collagen fibril may be regarded as an expression of the density of the intermolecular
bonds, the light bands representing regions where the macromolecules are bonded
together laterally (Grant, Home & Cox, 1965; Grant, Cox & Home, 1967; Cox,
Grant & Home, 1967).
In general, irradiation of native rat tail tendon with electrons resulted in a progressive
396
R. A. Grant, R. W. Cox and C. M. Kent.
loss of contrast in the bands. At the highest dose levels the bands completely
disappeared and the fibrils presented a very swollen appearance. The fibres irradiated in the dry state showed more severe changes than those irradiated in the presence
of water (Figs. n-14). In agreement with Bailey & Tromans (1964), the loss of band
structure may be regarded as being due to the penetration of negative stain solution
into the fibrils. This tendency of the molecules to separate may be tentatively explained as follows: (a) rupture of inter- and intramolecular hydrogen bonds leading to
disorganization of the specific bonding regions in the tropocollagen macromolecules;
and (b) scission of the polypeptide back-bone chains of the tropocollagen triple helix
resulting in fragmentation of the macromolecule.
The decreased solubility in acetic acid found in native rat tail tendon irradiated
in the wet state may be interpreted as being due to the formation of covalent, intermolecular cross-links. These probably result from random reactions of free radicals
arising from scission of the main polypeptide chains and do not produce an ordered
change that is visible with the electron microscope. This finding may be contrasted with
the ordered formation of covalent intermolecular cross-links when native collagen is
treated with glutaraldehyde resulting in well defined changes in the band pattern and a
general increase in the size of the light (A) bands (Grant et al. 1967).
In the case of rat tail collagen cross-linked with glutaraldehyde prior to irradiation,
the band pattern was not destroyed even with the highest dose (100 x io 4 J kg"1). A
contraction of the repeat period was noted, the B (dark) bands appearing to contract
more than the A bands. Since treatment with glutaraldehyde increases the size and
density of the A-band region we may infer that the intermolecular bonds, in the main
bonding zones, are reinforced by strong intermolecular bonds formed by the reaction
of glutaraldehyde with lysine side chains (Grant et al. 1967). These latter bonds,
being of covalent nature, are probably more resistant to rupture by radiation than
hydrogen or electrostatic bonds so that the band structure is well preserved in spite of
high doses of radiation. The part of the fibril structure represented by the B bands does
not appear to be stabilized to the same extent and its contraction may be interpreted as
being due to collapse of the triple helix structure of the macromolecules in these areas.
A similar disproportionate shrinkage of the A and B bands was found after heat
treatment of cross-linked collagen fibres (Grant et al. 1967).
Collagenase
Native rat tail tendon is rapidly digested by bacterial collagenase and is resistant to
trypsin and elastase. Irradiation of wet tendon with doses greater than 25 x io 4 J kg"1
resulted in increased resistance to bacterial collagenase, and with the highest dose no
digestion was found even after 5 days' exposure to the enzyme. On the other hand,
fibrils irradiated dry did not become resistant to the enzyme even at the highest dose
level. Bailey et al. (1964) showed that electron irradiation of rat tail tendon in the wet
state produced many intermolecular cross-links whereas, in the dry state, the main
effect was considered to be scission of polypeptide chains. We may thus interpret the
increased resistance of the wet-irradiated tendon to bacterial collagenase as being due
Electron irradiation of collagen
397
to the presence of intermolecular cross-links. This is supported by the fact that tendon
cross-linked with glutaraldehyde is completely resistant to this enzyme.
The results also suggest that irradiation of dry cross-linked collagen with electrons
leads to the breaking of certain bonds which, in the original state, hinder the action of
the enzyme. In the case of cross-linked collagen irradiated wet, it may be concluded
that, in addition to polypeptide chain rupture, new intermolecular bonds are formed
and that, above a certain critical dose level of about 50 x io4 J kg"1, sufficient of
these new cross-links are formed to neutralize the effect of main polypeptide chain
scission which appears to render the cross-linked collagen digestible by collagenase.
Autoclaving with water increased the rate of digestion of irradiated glutaraldehydetreated collagen by collagenase. This may be interpreted on the assumption that
autoclaving produces changes in the configuration of the polypeptide chains which
counteract the collagenase-inhibiting effect of glutaraldehyde- (Grant, 1965) and irradiation-induced cross-links. However, autoclaving was without effect on the material
that was subjected to the highest dose in the wet state and which was, presumably,
the most highly cross-linked of all the specimens.
Elastase
Native rat tail tendon is resistant to elastase. All the specimens irradiated in the
range 25-100 x io 4 J kg"1 were digested by elastase irrespective of whether the tendon
was in the wet or dry state. This seems to indicate that the introduction of cross-links
by electron bombardment in the wet state does not affect the processes whereby the
collagen is rendered susceptible to the action of elastase. This induced susceptibility
is probably due to changes in the configuration of the polypeptide chains resulting
from chain scission. It may be recalled that with collagenase irradiation of collagen in
the wet, but not the dry, state leads to increased resistance to enzymic attack. In this
connexion it may be noted that elastin, the natural substrate for elastase, is a highly
cross-linked protein.
Collagen which had been cross-linked with glutaraldehyde was also completely
resistant to elastase. Changes were noted after irradiation and, depending on whether
the material was treated wet or dry, the results contrasted sharply. At the lowest dose
of 25 x io4 J kg"1 the wet-irradiated collagen was completely digested, whereas the
tendon irradiated dry was resistant, while at 100 x io4 J kg"1 the reverse was found. It
may be concluded that progressive fragmentation of the polypeptide chains produced
by irradiation in the dry state results in increasing sensitivity of the cross-linked collagen to elastase. On the other hand, whereas the wet-irradiated collagen became
susceptible to elastase at 25 x io4 J kg"1, the formation of new cross-links by irradiation in the presence of water appears to result in the cross-linked collagen again
becoming resistant to elastase at the higher dose levels.
It has been found previously that autoclaving with water results in glutaraldehyde
cross-linked collagen becoming sensitive to elastase (Grant, 1965). A similar effect was
noted with the dry-irradiated cross-linked tendon which after autoclaving was completely digested by elastase within 24 h; the rate of attack on the wet-irradiated
collagen was also increased. No effect was noted on the glutaraldehyde-treated collagen
398
R. A. Grant, R. W. Cox and C. M. Kent
irradiated wet at ioo x io4 J kg"1; it thus seems that this material is so highly crosslinked that changes in configuration resulting from autoclaving do not render it
sensitive to elastase, unlike the unirradiated material.
Thus the effects of electron irradiation on the sensitivity of native and glutaraldehyde
cross-linked collagen to enzymes may be interpreted on the basis that irradiation in the
wet state results in chain scission plus the formation of intermolecular cross-links
while irradiation of the dry tendon results only in fragmentation of the polypeptide
chains. It may be assumed that both of these processes are accompanied by changes in
the configuration of the polypeptide chains.
Histological staining reactions
Irradiation of rat tail tendon with high doses of electrons resulted in the material
reacting abnormally with various histological stains. It seems unlikely that these
changes in tinctorial properties result from the formation of intermolecular crosslinks since glutaraldehyde cross-linked collagen stains in the usual manner unless
subjected to further degradation. Heating collagen in the wet state results in it tending
to stain like elastin and it would appear that a likely cause of the changed staining properties is an altered configuration of the polypeptide chains in the protein. Scission
of polypeptide chains appears to result from irradiation with electrons under both wet
and dry conditions. This may result in the triple helix arrangement of the chains, in
parts at least of the structure, becoming disorganized. As a result the collagen may
present a more non-polar surface and tend to react with histological stains like elastin
which has a very high content of amino acids with non-polar side chains. This view
that electron-irradiated collagen presents a more non-polar surface than the original
material is supported, to some extent, by the finding of Fullmer & Lillie (1956) that
acetylation or benzoylation of collagen caused it to react with elastic tissue stains. It
may be noted that heating collagen in the wet state at temperatures of 70-100 °C
results in it staining like elastin whereas dry collagen heated to the same temperature
shows little change in staining properties. In contrast, irradiation with electrons
produced marked changes in tinctorial reactivity regardless of whether the collagen
was irradiated wet or dry. Hence it may be concluded that the structural changes
induced by irradiation occur by a different mechanism to heat denaturation and there
is some evidence (R. A. Grant, unpublished observations) that loss of hydrogen is
involved. It is suggested that the altered histological staining properties of electronirradiated collagen result from changes in the spatial configuration of the polypeptide
chains in part at least of the structure.
These structural changes may result partly from main chain scission at various
points and partly from other causes, for example loss of hydrogen which cannot be
specified exactly at this stage.
Solubility changes
It was found by Bailey et al. (1964) that electron irradiation of collagen in the wet
state decreased its solubility in hot water (80 °C for 2 h) whereas irradiation in the dry
state increased its solubility. In the present study more drastic conditions were
Electron irradiation of collagen
399
employed. All the specimens of irradiated, native, rat tail tendon were soluble when
subjected to autoclaving with water at 103-4 kN m~2 (15 lb in.~2) pressure, with the
exception of the specimen which received iooxio 4 jkg~ 1 in the wet state. This
result indicated that the latter specimen was cross-linked as effectively as glutaraldehyde-treated collagen and that the cross-links were stronger than those present in
formaldehyde-tanned collagen which dissolves when autoclaved with water. In the
case of the dry-irradiated native specimens these were completely soluble when autoclaved with water, indicating that effective cross-linking was absent.
The results for the solubility in hot dilute alkali (Table 1) of native rat tail tendon are
in general conformity with the views of Bailey et al. (1964) that under wet conditions
both cross-linking and chain scission occur but that under dry conditions chain
scission is the predominating effect. In the case of the glutaraldehyde-tanned collagen,
irradiation had only a slight effect on the solubility in hot alkali and we may infer that
the cross-links initially present in this material are not broken down to any extent by
subsequent exposure to high energy electrons.
All the specimens of wet-irradiated native tendon proved to be insoluble in dilute
acetic acid at room temperature whereas the fibres irradiated dry showed marked
increases in the rate of dissolution. We may infer from this that sufficient cross-links
are introduced by irradiation in the wet state to produce insolubility under these
conditions, while fragmentation of the molecules resulting from dry irradiation has an
opposite effect. This result is in conformity with the finding of Bowes & Moss (1962)
for gamma-irradiated oxhide collagen which when irradiated in the dry state became
soluble in dilute acetic acid.
Amino acid analyses
In general no very marked differences in amino acid composition were noted between
the irradiated and control specimens. Hence it is not possible to ascribe any of the
observed effects of electron irradiation to the destruction of any individual or group
of amino acids.
The authors wish to thank Mr J. Bounden for assistance with the irradiation experiments,
and the Radiotherapy Department of St. Bartholomew's Hospital for making available the
linear accelerator. We also wish to thank Miss Anne Carter for histological preparations and
Mrs P. M. Tegerdine for assistance with photography.
REFERENCES
A. J., BENDALL, J. R. & RHODES, D. N. (1962). The effect of irradiation on the shrinkage temperature of collagen. Int. J. appl. Radiat. Isotopes 13, 131-136.
BAILEY, A. J., RHODES, D. N. & CATER, C. W. (1964). Irradiation-induced crosslinking of
collagen. Radiat. Res. 22, 606-621.
BAILEY, A. J. & TROMANS, W. J. (1964). Effects of ionizing radiation on the ultrastructure of
collagen fibrils. Radiat. Res. 23, 145-155.
BOWES, J. H. & Moss, J. A. (1962). The effect of gamma radiation on collagen. Radiat. Res. 16,
BAILEY,
211-223.
R. (1961). The effect of electron radiation on the tensile strength of tendon. Int. J.
Radiat. Biol. 4, 27-31.
BRAAMS, R. (1963). The effect of electron radiation on the tensile strength of tendon II. Int. J.
Radiat. Biol. 7, 29-39.
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S. & HORNE, R. W. (1959). A negative staining method for high resolution electron
microscopy of viruses. Biochim. biophys. Ada 34, 103-110.
Cox, R. W., GRANT, R. A. & HORNE, R. W. (1967). The structure and assembly of collagen
fibrils. I. Native collagen fibrils and their formation from tropocollagen. JlR. microsc. Soc. 87,
BRENNER,
123-142.
Cox, R. W. & HORNE, R. W. (1968). Accurate calibration of the magnification of the A.E.I.,
E.M. 6B/2 electron microscope using catalase crystals. Proc. 4th Europ. Conf. Electron
Microsc. vol. 1 (ed. S. Bocciarelli), p. 579. Rome: Tipographia Poliglotta Vaticana.
ELBERS, P. F. & PIETERS; J. (1964). Accurate determination of magnification in the electron
microscope. J. Ultrastruct. Res. n , 25-32.
FULLMER, H. M. & LILLIE, R. D. (1956). Some aspects of the mechanism of orcein staining.
J. Histochem. Cytochem. 4, 64-68.
GRANT, R. A. (1965). Preparation of elastin-like material from collagen by crosslinking followed
by heat treatment. Biochem. J. 97, 5C-7C.
GRANT, R. A., Cox, R. W. & HORNE, R. W. (1967). The structure and assembly of collagen
fibrils. II. An electron microscope study of crosslinked collagen. Jl R. microsc. Soc. 87,
143-155GRANT, R. A., HORNE, R. W. & Cox, R. W. (1965). New model for the tropocollagen macromolecule and its mode of aggregation. Nature, Lond. 207, 822-824.
KUNTZ, E. & WHITE, E. (1961). Effects of electron beam irradiation on collagen. Fedn Proc.
Fedn Am. Socs exp. Biol. 20, 376.
LEWIS, V. J., WILLIAMS, D. E. & BRINK, N. G. (1956). Pancreatic elastase: purification properties and function, jf. biol. Chem. 222, 705-720.
PERRON, R. R. & WRIGHT, B. A. (1950). Alteration of collagen structure by electron irradiation.
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{Received 19 December 1969)
Electron irradiation of collagen
All preparations have been negatively stained with potassium
phosphotungstate and all markers represent o-i fim.
Fig. 9. Native rat tail collagen. Alternating light (A) and dark (B) bands are prominent
and individual tropocollagen macromolecules are distinguishable. The period is
approximately 64 nm.
Fig. 10. Native rat tail collagen cross-linked with glutaraldehyde. The light (A) band
has increased at the expense of the dark (B) band. A well-defined light striation has
appeared in the B band.
401
402
R. A. Grant, R. W. Cox and C. M. Kent
Fig. I I . Native collagen irradiated in the dry state with 25 x io4 J kg"1. Crossstriations are no longer visible, although the fibrous appearance is still preserved.
Fig. 12. Native collagen irradiated in the dry state with 100 x io4 J kg"1. The material
is amorphous and shows no trace of cross-striations.
Electron irradiation of collagen
403
Fig. 13. Native collagen irradiated in the wet state with 50 x io 4 j kg"1. The fibril shows
areas of local swelling with loss of cross-striations. The intervening zone, however,
still shows obvious cross-striations.
Fig. 14. Native collagen irradiated in the wet state with 100 x io4 J kg""1. The shape of
the fibril is still preserved and there is a suggestion of cross-striation.
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R. A. Grant, R. W. Cox and C. M. Kent
Fig. 15. Cross-linked native collagen irradiated in the wet state with 25 x io4 J kg"1.
Individual tropocollagen macromolecules of approximately i-5 nm diameter are visible
and the alternating light (A) and dark (B) bands with a period of approximately 64 nm
are prominent. There is also a characteristic light cross-striation within each B band.
The irradiation has apparently had no effect on the morphology.
Fig. 16. Cross-linked native collagen irradiated in the wet state with 50 x io 4 j kg"1.
There is localized swelling and some loss of cross-striations. In other areas the
cross-striations are preserved.
Fig. 17. Cross-linked native collagen irradiated in the wet state with 100 x io 4 J kg"1.
Although the cross-striations were still plainly visible the normal period of the fibril
has been shortened, the B bands being more affected than the A bands.
Electron irradiation of collagen
Fig. 18. Cross-linked native collagen irradiated in the dry state with 50 x io 4 j kg"1.
The shape of the fibril has been preserved but the cross-striations, although present,
have been badly affected.
Fig. 19. Cross-linked native collagen irradiated in the dry state with 100 x io4 J kg"1.
The upper fibril is swollen and cross-striations are poorly seen. The lower fibril,
however, shows much better defined cross-striations.
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