THE EFFECTS OF IRRADIATION WITH HIGH ENERGY ...Irradiation altere thde reactivity of both nativ ane...

J. Cell Sci. 7, 387-405 (1970) 387Printed in Great Britain

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. KENTAgricultural Research Council, Institute of Animal Physiology, Babraham, Cambridge,England

SUMMARY

Native and cross-linked rat tail tendon collagen was irradiated with high energy electrons upto a maximum dose of 100 x io4 J kg"1 (100 Mrd). Fibres irradiated dry showed greater damagewhen examined in the electron microscope using negative staining techniques than thoseirradiated wet. Cross-linking with glutaraldehyde prior to irradiation resulted in the bandstructure being preserved even at the highest dose but an unequal shrinkage of the bands wasnoted.

Irradiation altered the reactivity of both native and cross-linked collagen with collagenaseand 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 ofradiation; different results were obtained after irradiation in the wet and dry states.

Irradiated collagen reacted abnormally with various histological stains and tended to resembleelastin 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 isaccompanied by the formation of intermolecular bonds. It also appears that changes in theconfiguration of the polypeptide chains accompany both these processes.

INTRODUCTION

In recent years a number of investigations have been made on the effects of ionizingradiation on various proteins, including collagen.

Although collagen is distributed throughout the body, it is usually present in smalland variable amounts in most tissues. Consequently, studies on the effects of irradia-tion on collagen have usually been made on tendon where it occurs in a relativelypure state.

Bailey, Bendall & Rhodes (1962) studied the effect of irradiation with 2 MeVelectrons on the shrinkage temperature of rat tail tendon collagen. Doses up to4oxio4jkg~1 (40 Mrd) were given at o °C. It was found that the hydrothermalshrinkage temperature decreased progressively with dose while the amount by whichthe fibres shortened under a fixed load increased from 6% for native collagen to a

25 C E L 7

388 R. A. Grant, R. W. Cox and C. M. Kent

maximum of 70% for collagen treated with doses greater than 6 x io4 J kg"1. Irradia-tion under various conditions led to the conclusion that these effects were due to thedirect action of the radiation on the protein molecule and not to subsequent chemicalattack by radicals from water.

Bailey & Tromans (1964) investigated the effects of electron radiation on the ultra-structure of collagen fibrils using the electron microscope and a negative stainingtechnique. Below iox io 4 jkg - 1 , no irradiation-induced effects were observed buthigher doses resulted in a gradual loss of the band contrast of the fibrils, and at40 x io4 J kg"1 the cross-banding had completely disappeared. The effects of irradia-tion on the fibrils were compared with those of other treatments such as the action ofurea and various cross-linking chemicals. It was concluded that loss of band patternwas primarily due to swelling of the fibrils resulting from a structural disorganizationof the collagen macromolecule and the subsequent uptake of water. Bailey, Rhodes &Cater (1964) found that intermolecular cross-links were produced when rat tailtendon collagen was irradiated in the presence of water, whereas, in contrast, irradia-tion in the dry state resulted in fragmentation of the macromolecules; no histologicalor enzymic observations were made on the irradiated collagen.

A detailed examination of the effects of electron radiation on the tensile strength ofbovine tendon was made by Braams (1961, 1963). He found that for dry tendon thedose required to reduce the tensile strength to 37% of its original value was approxi-mately i 8 x i o 4 j kg"1. For hydrated tendon, reduction of tensile strength by thesame amount required 46 x io4 J kg"1.

Other less detailed studies have been made of the effects of electron irradiation oncollagen. Perron & Wright (1950) found that changes in the physical appearance ofkangaroo tail tendon were less marked in tendon irradiated dry rather than wet.However, when the dry-irradiated material was subsequently immersed in watermost of the material dissolved, leaving only a thin gelatinous film that gave an amor-phous low angle X-ray diffraction pattern.

Kuntz & White (1961) reported that cross-linking predominated over chain scissionduring irradiation with electrons of collagen in the wet state.

The object of the present communication was to extend previous studies of theeffects of electron irradiation on collagen by (1) using a wider dose range, (2) includingcross-linked in addition to native collagen and (3) investigating changes in the histo-logical staining reactions of the irradiated collagen and its reactivity with collagenaseand elastase.

MATERIALS AND METHODS

Collagen preparations

Freshly dissected rat tail tendons were washed in cold saline (0-9 % NaCl) and stored in coldsaline until required. Hydroxyproline analysis showed that the fibres consisted of almost purecollagen. Fibres to be irradiated in the dry state were dried under vacuum in a desiccatorbefore being irradiated in narrow, stoppered glass tubes; the material to be irradiated wet wasplaced in tubes containing saline. The sample tubes were suspended in a beaker containing iceand 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 % glutaralde-hyde solution overnight; the fibres were then carefully washed and part of the preparation driedin vacuo.

Irradiation

The beaker containing the specimen tubes was placed directly in the beam from a 14 MeVlinear 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 atthe 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 speci-mens could not be measured in this way, but there was no evidence that these reached a highertemperature 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 alsoincubated 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 over-night at 4 °C followed by removal of elastin by centrifuging. The degree of digestion wasestimated by visual inspection.

Solubility

Samples of irradiated and control rat tail tendon were placed in 0-5 % acetic acid and allowedto stand overnight at room temperature. Solubility in hot water was tested by heating samples oftendon fibres with water in an autoclave at 103-4 kN m~2 (15 lb in.~2) pressure for 6 h. Furthersamples were heated at 95 °C in O'i N NaOH solution until dissolved and the times requiredfor complete solution recorded.

Histology

Specimens of irradiated and control rat tail tendon were fixed in 1 o % formol saline, embeddedin paraffin and sectioned. Sections were stained with orcein, Van Gieson stain, phosphotungsticacid/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 hin sealed tubes in an autoclave at 1034 kN m~2 (15 lb in.~2) pressure. The resulting hydroly-sates were then analysed by ion exchange chromatography using a Technicon amino acidanalyser. Only the specimens treated with 100 x io4 J kg~L irradiation (wet and dry, native andcross-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 finesuspension. The finely dispersed material was negatively stained on grids with a solution of2 % 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 atan 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 astandard, allowances being made for the variables causing errors in magnification (Elbers &Pieters, 1964; Cox & Home, 1968).

25-2

39° R. A. Grant, R. W. Cox and C. M. Kent

RESULTS

Enzyme reactions

Native wet fibres (Figs, i, 2). Irradiation of collagen fibres in the wet state withdoses up to 25 x io4 J kg"1 did not affect sensitivity to bacterial collagenase, completedigestion of the material being obtained. At 50 x io4 J kg"1 no digestion was notedafter 24 h but after 5 days the sample was completely digested. With a dose of100 x io4 J kg"1 no digestion was obtained with collagenase even after 5 days. At a doselevel of 2 x io4 J kg"1 the treated collagen was not attacked by elastase but at doselevels above 25 x io4 J kg"1 it was completely digested by this enzyme.

100 -

75

•S 50

0 -

100 -

75

.9 50WtoCJ

Q

25

0 -

25 50 100

Dose of electrons (x104 J kg"1)(1 Mrd = 104J kg"1)

Fig. 1

25 50 100

Dose of electrons (x 104 J kg"1)(1 Mrd = 104J kg"1)

Fig. 2

Fig. 1. Effect of collagenase on irradiated native collagen at pH 70 , 37 °C for 24 h.O - - O, dry; • • , wet.Fig. 2. Effect of collagenase on irradiated native collagen at pH 70, 37 °C for 5 days.O - - O, dry; • • , wet.

Native fibres irradiated in the dry state (Figs. 1, 2). Irradiation with electrons even atthe highest dose of 100 x io4 J kg"1 of fibres in the dry state did not result in any lossof sensitivity of the collagen to bacterial collagenase, in contrast to the results obtainedin the wet state. However, all the treated samples were completely digested by elastaseexcept for those which received the lowest dose of 2 x io4 J kg"1, which were resistant.Thus, irradiation with electrons in the dry as well as in the hydrated state results incollagen becoming sensitive to elastase to which it is normally resistant.

Cross-linked fibres irradiated in the wet state (Figs. 3-6). Collagen cross-linked withglutaraldehyde is usually completely resistant to the action of bacterial collagenase.Irradiation with electrons in the wet state resulted in the material becoming sensitiveto the action of collagenase although digestion was very slow. No effect was noted


after 24 h but after 5 days the samples which had received 50 x io4 J kg"1 ofradiation were partially digested by the enzyme while the samples which had received25 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 withelastase. Those treated with 25 and 50 x io4 J kg"1 were partly digested after 24 h andcompletely digested after 5 days while the fibres receiving the highest dose were notattacked even after 5 days.

100

75

•g 50

25 -

0 - ?-

392

100 h

75

s 50(U

Q

25

R. A. Grant, R. W. Cox and C. M. Kent

100

75

- i25 50 100

Dose of electrons (x104 J kg"1)(1 Mrd = 104 J kg"1)

Fig. S

25 50 100Dose of electrons (x 10" J kg"')

(1 Mrd = 10" J kg"1)

Fig. 6

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 for5 days. A - - A, dry; A A, wet.

D

25 50 100Dose of electrons (x10"J kg"1)

(1 Mrd=104 J kg"1)

Fig. 7

25 50 100Dose of electrons (x 104 J kg"1)

(1 Mrd=104J 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.


the wet and dry state, samples which had received 25 x io4 J kg"1 were resistant to theaction of collagenase, they were completely digested within 24 h after autoclaving withwater under the conditions specified. At a level of 5oxio4jkg"1, autoclaving in-creased the rate of digestion by collagenase. The degree of digestion after 24 h wasabout the same for both wet- and dry-irradiated samples as was obtained after 5 dayswhere the specimen had not been autoclaved. However, autoclaving had no effect onthe specimens given 100 x io4 J kg"1 in the wet state, these being undigested evenafter 5 days.

Autoclaving with water markedly increased the rate of digestion by elastase of wet-irradiated, cross-linked collagen at the 25xio4Jkg"] level but had no apparenteffect on the same material treated with 100 x io4 J kg"1 which appeared unaffected.In the case of cross-linked collagen irradiated dry with 25 x io4 J kg"1, this was notattacked by elastase but, after autoclaving, was completely digested. With a dose of50 x io4 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 reactivitywith elastase, complete digestion being obtained in 24 h.

Histological staining reactions

In general, irradiation with electrons resulted in the collagen fibres staining abnor-mally. This was so even though the temperature was maintained at a low level (below20 °C) during irradiation. With irradiated doses above 25xio4Jkg~1 the treatedcollagen tended to resemble elastin in its tinctorial reactivity. This result was obtainedwith fibres irradiated in the dry as well as in the hydrated state. Fibres irradiated withdoses 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 treatedcollagen stain as intensely as elastin. Very marked effects were noted in the case of thephosphotungstic acid/haematoxylin stain, the treated fibres usually tending to assumea deep purple colour in contrast to the red staining of the control material. Withhaematoxylin and eosin the irradiated fibres were usually stained mauve, in contrast tothe light pink of the controls, and with Weigert's elastic stain the treated fibres tendedto resemble elastin rather than collagen. Irradiated fibres also stained abnormallywith Luxol fast blue, often appearing pale grey or mauve instead of the clear, deepblue of the controls.

Solubility

All the irradiated specimens of native collagen were soluble when autoclaved withwater at 103-4 kNm~2 (15 lb in."2) pressure except the specimen which receivediooxio4jkg~1 in the wet state. The irradiated cross-linked specimens were allinsoluble when autoclaved with water.

Solubilities in dilute sodium hydroxide solution and acetic acid are shown in Tables 1and 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 andcontrol samples with both native and cross-linked collagen. Collagen cross-linkedwith glutaraldehyde showed an 80% drop in the lysine and hydroxylysine contents.

Table 1. Solubility of native and cross-linked collagen, wet- and dry-irradiatedand non-irradiated fibres in o-i N NaOH at 95 °C

Treatment

Native fibres

Cross-linkedfibres

(WetI Dry/Wet\Dry

#>

25

time

X

in

IO4J

11*2

3 0 0

2 4 0

min

kg"1 50 x io4 J kg"1 100

> 1 1

2

3 0 0

195

for complete solution.

X I O 4

602

2 6 0

2 1 0

Jkg"1 Control

17

3 1 0

—

SJative fibres

Cross-linkedfibres 1

Wet

Dry

IWetDry

Slightlyswollen

C2 5 *

NN

N

C1 0

NN

N

C5NN

Table 2. Solubility of native and cross-linked collagen, wet- and dry-irradiatedand non-irradiated fibres in 0-5% acetic acid at room temperature

Treatment 25 x io4 J kg"1 50 x io4 J kg"1 100 x io4 J kg"1 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 increasingdose of radiation. At a dosage level of 25 x io4 J kg"1, collagen that had been irradiatedin the dry state was markedly affected (compare Figs. 9 and 11). Most of the fibrilshad lost their cross-striations. Careful search, however, revealed occasional traces ofcross-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 lengthsof 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 io4 J kg-1, cross-striations had completely disappeared fromcollagen that had been irradiated in the dry state. Much of the collagen did not evenhave a fibrous appearance; it was present as amorphous, irregular masses. Collagenthat had been irradiated at the same dosage level but in the wet state showed lessalteration (Fig. 13). The changes varied from localized, swollen areas with loss of cross-striations, through generally swollen fibrils with perceptible but poorly defined cross-striations, to definable fibrils devoid of any cross-striations.


With dosages of 100 x io4 J kg"1 collagen that had been irradiated in the dry stateshowed neither formed fibrils nor any suggestion of cross-striations. The materialwas amorphous and irregular (Fig. 12). In collagen irradiated with the same dosage inthe wet state the changes were less severe (Fig. 14). Although amorphous andirregular masses of material were present the shape of the fibrils was mainlypreserved. Many of these fibrils possessed no cross-striations and were irregularlyswollen. In others traces of cross-striations remained.

Cross-linked collagen. The protection afforded by cross-linking with glutaraldehydeagainst the effects of irradiation was considerable. With dosages below 30 x io4 J kg"1

there was no change in the morphology of the collagen irradiated in either the wet orthe 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 collagenthat had been irradiated in the wet state showed some localized swelling of the fibrilswith relatively poor definition of the cross-striations in those areas (Fig. 16). Theremainder of a fibril might be unaffected. The collagen irradiated in the dry statewith 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 amore widespread decrease in the definition of the cross-striations than at 50 x io4 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 iooxio4 jkg"1 showed more markedchanges than collagen irradiated wet at the same dose levels. Swelling and loss ofdefinition of cross-striations were more widespread. Nevertheless, occasional fibrilswere remarkably well preserved (Fig. 19).

Although the differences between cross-linked collagen irradiated in the wet anddry 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 affectedmorphologically than that irradiated in the presence of water at the same dosage.

To illustrate the difference between native and cross-linked collagen, it may benoted that the changes in native collagen that had been irradiated in the wet state atdosages of 32 x io4 J kg"1 were always greater than those in cross-linked native collagenthat 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 thinfilaments of approximately 1-5 nm diameter running roughly parallel to the directionof the intact fibril. The alternating sequence of light (A) and dark (B) bands along thecollagen fibril may be regarded as an expression of the density of the intermolecularbonds, the light bands representing regions where the macromolecules are bondedtogether 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 completelydisappeared and the fibrils presented a very swollen appearance. The fibres irradi-ated in the dry state showed more severe changes than those irradiated in the presenceof water (Figs. n-14). In agreement with Bailey & Tromans (1964), the loss of bandstructure may be regarded as being due to the penetration of negative stain solutioninto the fibrils. This tendency of the molecules to separate may be tentatively ex-plained as follows: (a) rupture of inter- and intramolecular hydrogen bonds leading todisorganization of the specific bonding regions in the tropocollagen macromolecules;and (b) scission of the polypeptide back-bone chains of the tropocollagen triple helixresulting in fragmentation of the macromolecule.

The decreased solubility in acetic acid found in native rat tail tendon irradiatedin the wet state may be interpreted as being due to the formation of covalent, inter-molecular cross-links. These probably result from random reactions of free radicalsarising from scission of the main polypeptide chains and do not produce an orderedchange that is visible with the electron microscope. This finding may be contrasted withthe ordered formation of covalent intermolecular cross-links when native collagen istreated with glutaraldehyde resulting in well defined changes in the band pattern and ageneral 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 io4 J kg"1). Acontraction of the repeat period was noted, the B (dark) bands appearing to contractmore than the A bands. Since treatment with glutaraldehyde increases the size anddensity of the A-band region we may infer that the intermolecular bonds, in the mainbonding zones, are reinforced by strong intermolecular bonds formed by the reactionof glutaraldehyde with lysine side chains (Grant et al. 1967). These latter bonds,being of covalent nature, are probably more resistant to rupture by radiation thanhydrogen or electrostatic bonds so that the band structure is well preserved in spite ofhigh doses of radiation. The part of the fibril structure represented by the B bands doesnot appear to be stabilized to the same extent and its contraction may be interpreted asbeing 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 heattreatment of cross-linked collagen fibres (Grant et al. 1967).

Collagenase

Native rat tail tendon is rapidly digested by bacterial collagenase and is resistant totrypsin and elastase. Irradiation of wet tendon with doses greater than 25 x io4 J kg"1

resulted in increased resistance to bacterial collagenase, and with the highest dose nodigestion 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 doselevel. Bailey et al. (1964) showed that electron irradiation of rat tail tendon in the wetstate produced many intermolecular cross-links whereas, in the dry state, the maineffect was considered to be scission of polypeptide chains. We may thus interpret theincreased resistance of the wet-irradiated tendon to bacterial collagenase as being due


to the presence of intermolecular cross-links. This is supported by the fact that tendoncross-linked with glutaraldehyde is completely resistant to this enzyme.

The results also suggest that irradiation of dry cross-linked collagen with electronsleads to the breaking of certain bonds which, in the original state, hinder the action ofthe enzyme. In the case of cross-linked collagen irradiated wet, it may be concludedthat, in addition to polypeptide chain rupture, new intermolecular bonds are formedand that, above a certain critical dose level of about 50 x io4 J kg"1, sufficient ofthese new cross-links are formed to neutralize the effect of main polypeptide chainscission which appears to render the cross-linked collagen digestible by collagenase.

Autoclaving with water increased the rate of digestion of irradiated glutaraldehyde-treated collagen by collagenase. This may be interpreted on the assumption thatautoclaving produces changes in the configuration of the polypeptide chains whichcounteract the collagenase-inhibiting effect of glutaraldehyde- (Grant, 1965) and irra-diation-induced cross-links. However, autoclaving was without effect on the materialthat 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 therange 25-100 x io4 J kg"1 were digested by elastase irrespective of whether the tendonwas in the wet or dry state. This seems to indicate that the introduction of cross-linksby electron bombardment in the wet state does not affect the processes whereby thecollagen is rendered susceptible to the action of elastase. This induced susceptibilityis probably due to changes in the configuration of the polypeptide chains resultingfrom chain scission. It may be recalled that with collagenase irradiation of collagen inthe wet, but not the dry, state leads to increased resistance to enzymic attack. In thisconnexion it may be noted that elastin, the natural substrate for elastase, is a highlycross-linked protein.

Collagen which had been cross-linked with glutaraldehyde was also completelyresistant to elastase. Changes were noted after irradiation and, depending on whetherthe material was treated wet or dry, the results contrasted sharply. At the lowest doseof 25 x io4 J kg"1 the wet-irradiated collagen was completely digested, whereas thetendon irradiated dry was resistant, while at 100 x io4 J kg"1 the reverse was found. Itmay be concluded that progressive fragmentation of the polypeptide chains producedby irradiation in the dry state results in increasing sensitivity of the cross-linked col-lagen to elastase. On the other hand, whereas the wet-irradiated collagen becamesusceptible to elastase at 25 x io4 J kg"1, the formation of new cross-links by irradia-tion in the presence of water appears to result in the cross-linked collagen againbecoming resistant to elastase at the higher dose levels.

It has been found previously that autoclaving with water results in glutaraldehydecross-linked collagen becoming sensitive to elastase (Grant, 1965). A similar effect wasnoted with the dry-irradiated cross-linked tendon which after autoclaving was com-pletely digested by elastase within 24 h; the rate of attack on the wet-irradiatedcollagen was also increased. No effect was noted on the glutaraldehyde-treated collagen


irradiated wet at ioo x io4 J kg"1; it thus seems that this material is so highly cross-linked that changes in configuration resulting from autoclaving do not render itsensitive to elastase, unlike the unirradiated material.

Thus the effects of electron irradiation on the sensitivity of native and glutaraldehydecross-linked collagen to enzymes may be interpreted on the basis that irradiation in thewet state results in chain scission plus the formation of intermolecular cross-linkswhile irradiation of the dry tendon results only in fragmentation of the polypeptidechains. It may be assumed that both of these processes are accompanied by changes inthe configuration of the polypeptide chains.

Histological staining reactions

Irradiation of rat tail tendon with high doses of electrons resulted in the materialreacting abnormally with various histological stains. It seems unlikely that thesechanges in tinctorial properties result from the formation of intermolecular cross-links since glutaraldehyde cross-linked collagen stains in the usual manner unlesssubjected to further degradation. Heating collagen in the wet state results in it tendingto stain like elastin and it would appear that a likely cause of the changed staining pro-perties is an altered configuration of the polypeptide chains in the protein. Scissionof polypeptide chains appears to result from irradiation with electrons under both wetand dry conditions. This may result in the triple helix arrangement of the chains, inparts at least of the structure, becoming disorganized. As a result the collagen maypresent a more non-polar surface and tend to react with histological stains like elastinwhich has a very high content of amino acids with non-polar side chains. This viewthat electron-irradiated collagen presents a more non-polar surface than the originalmaterial is supported, to some extent, by the finding of Fullmer & Lillie (1956) thatacetylation or benzoylation of collagen caused it to react with elastic tissue stains. Itmay be noted that heating collagen in the wet state at temperatures of 70-100 °Cresults in it staining like elastin whereas dry collagen heated to the same temperatureshows little change in staining properties. In contrast, irradiation with electronsproduced marked changes in tinctorial reactivity regardless of whether the collagenwas irradiated wet or dry. Hence it may be concluded that the structural changesinduced by irradiation occur by a different mechanism to heat denaturation and thereis some evidence (R. A. Grant, unpublished observations) that loss of hydrogen isinvolved. It is suggested that the altered histological staining properties of electron-irradiated collagen result from changes in the spatial configuration of the polypeptidechains in part at least of the structure.

These structural changes may result partly from main chain scission at variouspoints and partly from other causes, for example loss of hydrogen which cannot bespecified exactly at this stage.

Solubility changes

It was found by Bailey et al. (1964) that electron irradiation of collagen in the wetstate decreased its solubility in hot water (80 °C for 2 h) whereas irradiation in the drystate increased its solubility. In the present study more drastic conditions were


employed. All the specimens of irradiated, native, rat tail tendon were soluble whensubjected to autoclaving with water at 103-4 kN m~2 (15 lb in.~2) pressure, with theexception of the specimen which received iooxio4jkg~1 in the wet state. Thisresult indicated that the latter specimen was cross-linked as effectively as glutaral-dehyde-treated collagen and that the cross-links were stronger than those present informaldehyde-tanned collagen which dissolves when autoclaved with water. In thecase of the dry-irradiated native specimens these were completely soluble when auto-claved 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 arein general conformity with the views of Bailey et al. (1964) that under wet conditionsboth cross-linking and chain scission occur but that under dry conditions chainscission 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 thatthe cross-links initially present in this material are not broken down to any extent bysubsequent exposure to high energy electrons.

All the specimens of wet-irradiated native tendon proved to be insoluble in diluteacetic acid at room temperature whereas the fibres irradiated dry showed markedincreases in the rate of dissolution. We may infer from this that sufficient cross-linksare introduced by irradiation in the wet state to produce insolubility under theseconditions, while fragmentation of the molecules resulting from dry irradiation has anopposite 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 becamesoluble in dilute acetic acid.

Amino acid analyses

In general no very marked differences in amino acid composition were noted betweenthe irradiated and control specimens. Hence it is not possible to ascribe any of theobserved effects of electron irradiation to the destruction of any individual or groupof 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 thelinear accelerator. We also wish to thank Miss Anne Carter for histological preparations andMrs P. M. Tegerdine for assistance with photography.

REFERENCES

BAILEY, A. J., BENDALL, J. R. & RHODES, D. N. (1962). The effect of irradiation on the shrink-age temperature of collagen. Int. J. appl. Radiat. Isotopes 13, 131-136.

BAILEY, A. J., RHODES, D. N. & CATER, C. W. (1964). Irradiation-induced crosslinking ofcollagen. Radiat. Res. 22, 606-621.

BAILEY, A. J. & TROMANS, W. J. (1964). Effects of ionizing radiation on the ultrastructure ofcollagen fibrils. Radiat. Res. 23, 145-155.

BOWES, J. H. & Moss, J. A. (1962). The effect of gamma radiation on collagen. Radiat. Res. 16,211-223.

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{Received 19 December 1969)


All preparations have been negatively stained with potassiumphosphotungstate and all markers represent o-i fim.

Fig. 9. Native rat tail collagen. Alternating light (A) and dark (B) bands are prominentand individual tropocollagen macromolecules are distinguishable. The period isapproximately 64 nm.Fig. 10. Native rat tail collagen cross-linked with glutaraldehyde. The light (A) bandhas increased at the expense of the dark (B) band. A well-defined light striation hasappeared in the B band.


Fig. I I . Native collagen irradiated in the dry state with 25 x io4 J kg"1. Cross-striations 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 materialis amorphous and shows no trace of cross-striations.


Fig. 13. Native collagen irradiated in the wet state with 50 x io4j kg"1. The fibril showsareas 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 ofthe fibril is still preserved and there is a suggestion of cross-striation.

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4°4 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 visibleand the alternating light (A) and dark (B) bands with a period of approximately 64 nmare 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 io4j kg"1.There is localized swelling and some loss of cross-striations. In other areas thecross-striations are preserved.Fig. 17. Cross-linked native collagen irradiated in the wet state with 100 x io4 J kg"1.Although the cross-striations were still plainly visible the normal period of the fibrilhas been shortened, the B bands being more affected than the A bands.

Electron irradiation of collagen 4°5

Fig. 18. Cross-linked native collagen irradiated in the dry state with 50 x io4j 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.

THE EFFECTS OF IRRADIATION WITH HIGH ENERGY ...Irradiation altere thde reactivity of both nativ ane...

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