Biochem. J. (1975) 145, 287-297Printed in Great Britain

Isolation and Chemical Characterization of Collagen inBovine Pulmonary Tissues

By GILLIAN FRANCIS and JOHN THOMASDepartment ofBiochemistry, University College, P.O. Box 78, CardiffCF1 1XL, U.K.

(Received 23 July 1974)

1. The contents of the fibrous proteins collagen and elastin in the pleural and parenchymalregions of bovine lungs were determined. The collagen content was approx. 70% (g/100gof salt-extracted defatted powder) in each tissue, and the elastin content was 28% in pleuraand 13.5% in parenchyma. 2. Purification of the insoluble collagen from the pleura andparenchyma of bovine lungs by various methods was attempted. The collagen fractionsisolated after incubation ofthe pulnonary tissues with the proteolytic enzymes collagenase('collagenase-soluble' fraction) or pancreatic elastase ('elastase-insoluble' fraction) eachcontained approx. 87% of the total collagen initially present. 3. Both collagen fractionswere chemically analysed for their amino acid and carbohydrate contents and were foundto be similar to those of the intact interstitial collagens isolated from skin, bone andtendon. 4. The contents of the two aldimine cross-linking compounds, dehydrohydroxy-lysinonorleucine and dehydrodihydroxylysinonorleucine, were determined in the bovinepulmonary collagen fractions, and were found to decrease with increasing age of theanimals, and were similar to the values found in intact collagens from bone and tendon.

Previous work from this laboratory on the fibrousproteins in lung has been concerned with elastin.John & Thomas (1971) reported that most of theelastin present in whole bovine lung was concentratedin the pleural and parenchymal regions. These pulmo-nary elastins had chemical compositions similar tothe well-characterized elastins isolated from aorta andligamentum nuchae of the same animals. John &Thomas (1972) also examined the effect ofagingon thechemical compositions of elastins isolated from theaortas and pulmonary tissues of human subjects.During these various studies it became apparent thatthe pleura and parenchyma of lungs also containsubstantial amounts ofcollagen and structural glyco-proteins, and the present paper and that of Francis& Thomas (1975) describe the isolation and chemicalcharacterization of these components from bovinelung.

MNterials and Metod

Materials

Elastase (pancreatopeptidase, EC 3.4.21.1 1) was

purchased from BDH Chemicals Ltd., Poole, Dorset,U.K. Collagenase EC 3.4.99.5; from Clostridiumhistolyticum, type III fraction 'A'), pepsin (EC3.4.23.1 ; 2 x crystallized) and proteinase (EC 3.4.24.4;from Streptomyces griseus, repurified type VI, Pro-nase) were from Sigma (London) Chemical Co., Lon-don S.W.6, U.K. Trypsin (EC 3.4.21.4; 2x crystal-lized, salt-free) and a-chymotrypsin (EC 3.4.21.1)

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were from Worthington Biochemical Corp., Free-hold, N.J., U.S.A. KB3H4 (3000mCi/mmol) wasobtained from The Radiochemical Centre, Amer.sham, Bucks., U.K. Pure samples of hydroxylysinoPnorleucine and dihydroxylysinonorleucine were sup.plied by Dr. A. J. Bailey (Agricultural ResearchCouncil, Meat Research Institute, Langford, Bristol,U.K.). Pure samples of desmosine, isodesmosine,lysinonorleucine and merodesmosine were isolatedfrom elastin from bovine ligamentum nuchae(Thomas et al., 1963; Francis et al., 1973). SephadexG-25 and G-10 were supplied by Pharmacia (G.B.)Ltd., London W.5, U.K. All other chemicals were ofanalytical grade and were supplied by BDH Chemi-cals Ltd., Poole, Dorset, U.K.

Initial treatment ofbovine pulmonary tissues

The visceral pleura and parenchyma were removedfrom the lungs of 3-year-old cattle, scraped free of alladhering tissues and washed repeatedly with ice-coldwater. The tissues were freeze-dried, cut into smallpieces, mixed with solid CO2 and powdered in ahamuner-mill. The resulting fine powders were sus-

pended in 1 M-NaCl (1: 500, w/v), stirred at 40C for24h, centrifuged at 15000g for 20min and the super-natant was discarded. Extraction with NaCl wasrepeated at least twice more and the residues werewashed with water until salt-free. They were then de-fatted by dispersion in chloroform-methanol (2:1,v/v) for 24h at 40C, filtered on a glass sinter withsuction, washed successively with ethanol, acetone

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G. FRANCIS AND J. THOMAS

and ether and finally dried in vacuo. The powderswere chemically analysed for their nitrogen, hydroxy-proline and carbohydrate contents and for theircollagen, elastin and glycoprotein contents.

Attempted solubilization of collagen from bovinepulmonary tissues

The salt-insoluble and defatted powdered residuesof pleura and parenchyma contained three groups offibrous proteins, namely collagen, elastin and struc-tural glycoproteins. Because of the insolubility of allthese proteins the isolation of collagen in a pure formposed a considerable problem. Several differentmethods were tried to solubilize selectively either thecollagen or the elastin and structural glycoproteins.These methods included treatments with concentratedsolutions of urea and guanidine hydrochloride, mildalkali, mild acid and various proteolytic enzymes ofdifferent specificities.

Portions (0.5g) of the powdered residues were sus-pended separately in 50ml volumes of 8M-urea, 5M-guanidine hydrochloride, 0.5 M-acetic acid and 0.1 M-NaOH and stirred at 40 and 40°C for 48 h. The sus-pensions were centrifuged at 150OOg for 20min, theinsoluble material was washed with water, and thewashings were combined with the respective super-natants and passed through a glass sinter under suc-tion. The soluble extracts were dialysed against water,concentrated on a rotary evaporator and freeze-dried.A further portion was suspended in 0.1 M-NaOH andincubated in a boiling-water bath for 45min by themethod of Lansing et al. (1952). The suspension wascentrifuged and the residue washed with water at98°C and dried with ethanol and acetone. The super-natant and washings were combined, the pH wasadjusted to pH7 with HCI and the solution stored atOOC.For the enzyme-digestion experiments, 1 g portions

ofthe original powdered residues were incubated withenzyme (10mg) as suspensions in 100ml volumes atthe appropriate pH. Pepsin was used in the presenceof 0.05% acetic acid, pH3.5, trypsin, chymotrypsinand Pronase were in 0.1 M-calcium acetate adjusted topH 8.0 with NH3, collagenase was in 1 mM-CaCI2adjusted to pH7.4 with NH3 and pancreatic elastasewas in 0.1 M-(NH4)2CO3, pH8.9. Mixtures weregently shaken at 40°C for 48h before centrifuging.Each residue was washed with 50ml of water and thewashings and supernatants were combined and con-centrated to about 20ml in a rotary evaporator at40°C. In those cases in which Ca2+ ions were presentthese were removed by titration with oxalic acid.Precipitated calcium oxalate was separated by centri-fugation and washed with small volumes of water.The supernatant and washings were combined andfreeze-dried.

All the freeze-dried soluble extracts obtained by thevarious treatments described above were weighed andtheir hydroxyproline contents determined as an indexof the presence of solubilized collagen.

'Collagenase-soluble' and 'elastase-insoluble' collagenfractions isolatedfrom bovine pulmonary tissues

Analysis of the soluble fractions obtained by thevarious treatments described above established thatthe ones obtained by incubation with collagenase(designated 'collagenase-soluble') contained thehighest content of hydroxyproline and hence thepurest collagen (i.e. partially degraded collagen). Incontrast, the soluble fractions obtained by treatmentwith elastase contained comparatively little hydroxy-proline, but the insoluble residues (designated 'elas-tase-insoluble') remaining after such treatment con-tained hydroxyproline (and hence collagen) inamounts similar to those noted for the 'collagenase-soluble' fractions. The yield of the 'collagenase-soluble' fractions was improved by pretreating theoriginal salt-extracted defatted residues with 5M-guanidine hydrochloride before incubation withcollagenase. The 'collagenase-soluble' and 'elastase-insoluble' fractions were then selected for detailedchemical analyses.

In addition to 3-year-old animals the 'collagenase-soluble' and 'elastase-insoluble' fractions were alsoprepared from the pulmonary tissues of 1-week-oldand 16-year-old animals.

Isolation ofglycopeptidesfrom the 'elastase-insoluble'collagen fraction from adult bovine pleuraThe 'elastase-insoluble' pleural fraction (4g) was

suspended in 150ml of 1 mM-CaCl2 adjusted to pH7.4with NH3 and was solubilized by digestion with col-lagenase (13mg) at 400C. After 36h the pH was raisedto 8.0 and digestion was continued by addition ofPronase (25mg) for 24h. The mixture was concen-trated to a volume of 15ml on a rotary evaporatorand separated on a Sephadex G-25 column(145cmx2.4cm) which was equilibrated and elutedwith 0.1 M-pyridine-acetate buffer, pH 5.7. Fractions(lOml) were collected and portions (0.05ml) testedwithninhydrinandanthrone reagentsbythemethod ofButler & Cunningham (1966). Two main peaks wereseen; the first peak contained glycopeptides and thesecond peak contained only peptides. The glyco-peptide-containing fractions were combined andfreeze-dried. A portion (200mg) was hydrolysed with2M-NaOH at 100°C for 24h under the conditions ofButler & Cunningham (1966). Under these circum-stances the material was degraded to a mixture ofglycopeptides and amino acids. The hydrolysate wasdesalted by passage through a small column

1975

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PULMONARY COLLAGEN

(lOcmxO.9cm) of Dowex 50 (H+ form) and washingthe column with water. The glycopeptides wereeluted with 2M-NH3. The eluate was concentrated ona rotary evaporator and the bulk of the free aminoacids were removed by separation on a SephadexG-10 column (75cmx 1.5cm) which was equilibratedand eluted with 0.1 M-pyridine-acetate buffer, pH 5.7.Fractions (2.5 ml) were collected and portions(0.05 ml) tested with ninhydrin and anthrone re-agents. Two main peaks were observed, the firstcontaining glycopeptides and the second aminoacids. The fractions containing glycopeptides werecombined and freeze-dried. The glycopeptides wereseparated from residual ninhydrin-positive materialsby preparative chromatography on washed Whatmanno. 1 paper in the solvent system butan-1-ol-aceticacid-water (4:1:5, by vol.) for 96h. The glycopeptidematerial migrated as two slow-moving ninhydrin-positive spots (A and B) which were eluted from thepaper with water and freeze-dried. When portionsof each were applied to a Locarte amino acid auto-analyser glycopeptide A was eluted in a position closeto a standard methionine sample and glycopeptide Bwas eluted in a position between standard samples oftyrosine and hydroxylysine. Hydrolysis of glyco-peptide A in 0.2M-HCI at 100°C for 4h led to itspartial conversion into glycopeptide B. Portions ofglycopeptides A and B were each hydrolysed with5.7M-HCI, after which hydroxylysine was identifiedand measured on the autoanalyser. Further portionswere subjected to methanolic-HCI hydrolysis beforeanalysis for galactose and glucose by g.l.c. Glyco-peptide A contained hydroxylysine, galactose andglucose in the molar proportions 1: 1.3:1.5. Glyco-peptide B contained hydroxylysine and galactose inthe molar ratio 1:1.3 and contained only traceamounts of glucose.

Preparation ofintact collagensfrom other tissue sources

Insoluble collagens from the tendons of the tailofa 3-month-old rat and from the Achilles tendon ofa1-week-old calf were isolated as follows. The tendonswere cut into small pieces, washed with ice-coldwater, homogenized in IM-NaCl (1: 200, v/w) in a

VirTis '45' homogenizer, centrifuged and the residueswashed with water. After defatting with organic sol-vents as described under 'Initial treatment of bovinepulmonary tissues' the residues were dried in vacuo.

This treatment yielded pure insoluble collagens fromthese tissue sources.The leg bones were dissected from 24 1-day-old

chicks and, after removal of the cartilaginous endsand the marrow, were scraped free of all other tissues.They were washed with cold water, homogenized in1 M-NaCl in a VirTis '45' homogenizer, washed free ofsalt and dried in vacuo. The powder was reduced with

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KB3H4 as described under 'Measurement ofreduciblecross-links in collagen' in order to stabilize collagencross-links before the lengthy decalcification process.Calcium was removed by stirring the reduced powder(1 g) with 0.5M-EDTA (lOOml, adjusted to pH7.4with NaOH) at 4°C for 6 days, the EDTA being re-newed each day. The insoluble material was recoveredby centrifugation, washed with water and defattedwith organic solvents as above. Insoluble collagencomprised 20% (w/w) of the bone. A section from thecentral shaft of the femur of a 3-year-old cow wastreated in the same way after being powdered withsolid CO2 in a hammer-mill and the insoluble col-lagen recovered was 18.5% of the bone.

'Collagenase-soluble' and 'elastase-insoluble' frac-tions were prepared from the intact collagens ofchick bones and of 1-week-old calf tendon by themethods described under 'Attempted solubilizationof collagen from bovine pulmonary tissue'.

Analytical methodsMeasurement of carbohydrate. Total carbohydrate

was determined by the anthrone method ofYemm &Willis (1954). Galactose and glucose were determinedby the g.l.c. method of Bhatti et al. (1970).Measurement of hydroxyproline. The method of

Stegemann & Stalder (1967) was used.Measurement of nitrogen. The method was based

on the use of Nessler reagent by Koch & McMeekin(1924).Amino acid analyses. Samples (10mg in 5ml of

constant-boiling HCI) were hydrolysed in evacu-ated sealed glass tubes. Collagen was hydrolysed for24h and elastin for 48h. Amino acid analyses wereperformed on a Locarte autoanalyser by the proce-dure of John & Thomas (1971).

Determination of elastin in salt-extracted defattedpowdered bovine pleura andparenchyma. The methodof Lansing et al. (1952) was used. The powders weresuspended in 0.1 M-NaOH (1:50, v/w) in boiling-water bath for 45min and the insoluble material(elastin) was recovered by filtration through sinteredglass, washed with water and after treatment withorganic solvents dried in vacuo. The elastins wereweighed and analysed.Measurement of reducible cross-links in collagent.

The cross-links measured were dehydrohydroxy-lysinonorleucine (Bailey & Peach, 1968) and dehydro-dihydroxylysinonorleucine (Mechanic & Tanzer,1970; Robins & Bailey, 1973). The structural formu-lae are shown in Scheme 1, the latter being shown inthe enol and keto forms. These compounds are acid-labile because they contain aldimine bonds. However,reduction of the collagen converts them into the acid-stable hydroxylysinonorleucine and dihydroxylysino-norleucine respectively, which can then be isolatedfrom acid hydrolysates of collagen on an auto-

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289

G. FRANCIS AND J. THOMAS

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anayser and measured with ninhydrin reagent Be-cause of the low content of these cross-links in col-lagen it was necessary to analyse 60mg amounts ofcollagen hydrolysates and to perform a preliminaryfractionation on a resin column followed by re-fractionation of those fractions that contained thecross-links. To facilitate the localization of the cross-links duringthe first fractionationproceduretheywereradioactively labelled by using KB3H4 in the pre-lininary reduction step.

Collagen (1 g) was supended in 0.05M-Na2CO3,pH7.4 (100mI), and stirred with 30mg of KB3H4(prepared by diluting radioactively labelled materialwith unlabelled KBH4 to give a mixture containing11 mCi/mmol of KB3H4) at room temnperature(190() for 1 h. Excess of KBH4 was destroyed byacidification with acetic acid to pH3.0, the mixturecentrifuged, the residue washed with water and driedwith organic solvents. Portions of the reduced col-lagen (120mg) were hydrolysed in constant-boilingHCI (20ml) for 24h at 1050(. After removal of HCIon a rotary evaporator, the hydrolysate was dissolvedin 0.2M-sodium citrate-HCl buffer, pH2.2, and frac-tionated on a column (26cm xO.9cm) of sulphonatedpolystyrene on a Locarte autoanalyser. The elutingbuffer system was 0.2M-sodium citrate-HI, pH4.25(for 90min), 0.35M-sodium citrate-HCI, pH5.28(for 120min) and I.OM-sodium citrate-HCa, pH6.65(for 125min). A fraction collector was attached to thebottom of the colunm and fractions (2.5ml; collectedevery 5min) were tested for radioactivity by addingportions (0.05ml) to lOml of scintillation liquid[O.6g of 1,4-bis-(4-methyl-5-phenyloxazol-2-yl)ben-zene, 7g of 2,5-diphenyloxazole in 750m1 of tolueneand 250ml of 2-methoxyethanol] and counting on aPackard Tri-Carb liquid-scintillation counter. 3H-labelled dihydroxylysinonorleucine mnerged in frac-tions 34-37. These fractions were combined, dilutedwith an equal volume of water and the pH was ad-justed to 2.2 with HCI. One-half of the volume wasthen re-fractionated on the same column but theautoanalyser and ninhydrin reagent were used toquantify the material. The eluting buffer system was0.2M-sodium citrate-Ha, pH4.25 (for 150min),0.35M-sodiumcitrate-HCI, pH 5.28 (for 105min) and1.0M-sodium citrate-HCI, pH6.65 (for 40min).

3H-labelaed hydroxylysinonorleucine emerged fromthe column during the preliminary fractionation infractions 39-42. These were combined, diluted,acidified and one-half of the volume was re-fractionated and analysed as described above exceptthat -the eluting buffer system was that used in thepreliminary column fractionation.

In the second fractionation procedure when nin-hydrin was used as colour reagent both the di-hydroxylysinonorieucine and hydroxylysinonor-leucine moved as double peaks. Separate experiments

1975

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PULMONARY COLLAGEN

showed that authentic preparations of these com-pounds also moved as double peaks and gave nin-hydrin colour yields of twice that of leucine whencalculated on a molar basis. The concentrations ofthese cross-links in collagen were expressed as resi-dues/tropocollagen molecule (300000g).

Detection and measurement of e-(y-glutamyl)lysineand e-(y-glutamyl)hydroxylysine cross-links in bovinepleural 'elastase-insoluble' collagen. The method ofdetection was that of Pisano et al. (1969), who ex-amined these cross-links in fibrin. The method con-sisted of cyanoethylation of the collagen followed byacid hydrolysis, a procedure under which any lysineor hydroxylysine residue involved in this type oflinkage would yield free lysine or hydroxylysine,whereas un-cross-linked lysine and hydroxylysinewould yield the N-carboxyethyl derivatives, whichwould be eluted in different positions from lysine andhydroxylysine on an autoanalyser.A portion (50mg) of pleural 'elastase-insoluble'

collagen, which had been digested with collagenaseand pronase (described above), was incubated withshaking in a mixture of 2ml of aq. 10% (v/v) triethyl-amine and 2ml of acrylonitrile in a stoppered tube at37°C for 9 days. The contents were evaporated todryness on a rotary evaporator and hydrolysed in

5.7M-HCI at 1050C for 24h in a sealed glass tube.After removal of HCl on a rotary evaporator thehydrolysate was dissolved in 0.2M-sodium citrate-HCl buffer, pH2.2, and fractionated on a column(26cm x 0.9cm) of sulphonated polystyrene resin on aLocarte autoanalyser. The eluting buffer was 0.35M-sodium citrate-HCI, pH5.28, and fractions were col-lected from the bottom of the column at 4min inter-vals. Those fractions eluted where standard lysineand hydroxylysine had previously been found to beeluted were combined separately, diluted with waterand their pH was adjusted to 2.2 with HCl. They werethen re-fractionated on the same column, beingeluted with 0.35M-sodium citrate-HCI, pH 5.28, andthe lysine and hydroxylysine were measured withninhydrin reagent.

Results

Analysis of initial salt-extracted defatted powderedbovine pulmonary tissues

The analyses for pleural and parenchymal powdersprepared from a 3-year-old animal are shown inTable 1. Elastin contains less than 2% (w/w) ofhydroxyproline and it was calculated that only a

Table 1. Analysis ofinitialsalt-extracted defattedpowdered bovinepleura andparenchymaThe results are expressed in g/100g of tissue powder. Glycoprotein concentrations were those reported by Francis &Thomas (1975).

Tissue source Nitrogen Carbohydrate Hydroxyproline CollagenPleura 16.6 0.94 9.76 68.0Parenchyma 16.3 4.10 9.10 72.0

Elastin Glycoproteins28.013.5

4.015.5

Table 2. Attemptedsolubilization ofcollagenfrom salt-extracteddefattedpleura andparenchyma of3-year-oldcattle

Results for parenchyma are in parentheses. For further details see the text.

Treatment8 M-Urea

5m-Guanidine hydrochloride

0.5M-Acetic acid

0.1 M-NaOH

CollagenaseChymotrypsinTrypsinPepsinPronasePancreatic elastase

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Time of treatment Temperature Material solubilized Hydroxyproline solubilized(h) (OC) (g/lOOg) (g/lOOg)

48 4 1.0 0.548 40 3.3 (4.2) 2.6 (0.7)48 4 1.5 1.048 40 6.5 (16.0) 1.9 (4.2)48 4 1.2 0.948 40 4.7 (1.4) 3.4 (0.2)48 4 1.0 (7.0) 0.7 (0.7)48 40 42.5 (39.5) 41.2 (31.5)0.75 98 71.9 (86.5) 95.0 (96.0)72 40 59.9 (61.0) 88.0 (87.0)72 40 10.8 (29.1) 6.4 (6.2)72 40 9.9 (31.7) 7.5 (7.6)72 40 46.4 (10.5) 39.8 (1.3)72 40 48.0 (51.5) 28.4 (20.5)18 40 38.2 (39.0) 10.0 (12.3)

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G. FRANCIS AND J. THOMAS

negligible amount (3-5 %) ofthe total hydroxyprolinein the powders was due to elastin. Since collagen isthe only other animal protein known to containhydroxyproline (about 13.5 %), the collagen presentin the powders could be determined by measuringhydroxyproline. It was established that the contentsof collagen in the pleural and parenchymal powderswere 68 and 72% respectively.

Attemptedsolubilization ofcollagenfrom bovinepulmo-nary tissues

The results of attempts to solubilize the collagenfrom the salt-extracted defatted powdered pleura andparenchyma prepared from 3-year-old animals arepresented in Table 2. Incubations at 4°C for 48h insolutions of concentrated urea or guanidine hydro-chloride, dilute acid or dilute alkali dissolved littlehydroxyproline-containing material (hence colla-gen). Apart from dilute alkali, incubations at 40°Cextracted only slightly more collagen and structuralglycoproteins. Incubations in 0.1 M-NaOH at 98°Cfor 45min dissolved all the collagen and structuralglycoproteins and left insoluble elastin.The results of digestion with proteolytic enzymes

(Table 2) can best be considered in the light of theobservations of Thomas & Partridge (1960) that pureelastin is not solubilized by treatments at 40°C withchymotrypsin, trypsin, pepsin or collagenase but issolubilized by pancreatic elastase and pronase.Bacterial collagenase has been shown by Mandl et al.(1964) to dissolve collagen, and the other proteinaseslisted above have been shown (e.g. by Drake et al.,1966) to have only limited solubilizing effects on col-lagen. Francis & Thomas (1975) have reported thatall the above proteinases other than collagenasesolubilized the structural glycoproteins present inpulmonary tissues.Table 2 shows that digestion of the salt-extracted

defatted pleura and parenchyma with each of theabove proteinases had various solubilizing effectson the hydroxyproline-containing material (hencecollagen). Collagenase solubilized about 87% of thetotal collagen present and did not affect elastin or thestructural glycoproteins. Chymotrypsin and trypsindissolved only small amounts of collagen but dis-solved the structural glycoproteins. Since pepsin hasno action on elastin the material solubilized by thisenzyme from pleura was a mixture ofcollagen (40% oftotal) and structural glycoproteins. In the case ofparenchyma, pepsin had little solubilizing effect oncollagen but dissolved part of the structural glyco-proteins. Pronase dissolved the elastin, the structuralglycoproteins and between 20 and 28% of total col-lagen in both tissues. Since pancreatic elastase isknown to dissolve elastin and structural glycopro-teins, and since only about 11 % of total collagen was

solubilized, most of the collagen remained in the in-soluble residue after treatment with this enzyme.

In summary, no single treatment was capable ofisolating the total collagen, either in a soluble de-graded form or in an insoluble intact form. However,the fractions obtained which seemed to possess thehighest concentrations of collagen (as judged fromhydroxyproline measurements) were the 'collagenase-soluble' and the 'elastase-insoluble' fractions. Thesefractions contained about 87% of the collagen initi-ally present. The collagen present in the 'elastase-insoluble' fraction was still only sparingly solublewhen stirred in solutions of 5M-guanidine hydro-chloride or in 0.5M-acetic acid at room temperaturefor 24h.

Chemical compositions ofthe 'collagenase-soluble' and'elastase-insoluble' collagen fractions isolated frombovine pulmonary tissues

Fractions prepared from salt-extracted defattedpleura and parenchyma of 3-year-old animals had theamino acid and carbohydrate compositions shownin Table 3. The main features of the composition arethe high contents of glycine, proline, alanine andhydroxyproline, the presence of hydroxylysine andthe low content of carbohydrate. These analyses arevery similar to those reported by Eastoe (1967) forthe fibrillar collagens isolated from bone, skin andtendon. The great bulk of the carbohydrate presentin the pulmonary fractions was identified as galactoseand glucose. Glycopeptides were isolated from thepleural 'elastase-insoluble' fraction which were simi-lar to those isolated by Butler & Cunningham (1966)and Cunningham & Ford (1968) from skin collagenand by Spiro (1969) from tendon collagen. Theseglycopeptides contained the monosaccharide galac-tose and the disaccharide glycosylgalactose, each ofwhich was linked by an O-glycosidic bond to thehydroxyl group of a hydroxylysine residue.The compositions of the 'collagenase-soluble' and

'elastase-insoluble' collagen fractions indicated thatthese fractions were substantially free from elastinand structural glycoproteins. However, the presenceof trace amounts of desmosine, isodesmosine, mero-desmosine and lysinonorleucine (known cross-linkingcompounds present in elastin) and galactosamineindicated slight contamination with elastin andstructural glycoproteins.

Contents of 'reducible cross-links' in 'collagenase-soluble' and 'elastase-insoluble' collagen fractions iso-latedfrom bovine pulmonary tissues

It was pointed out under 'Analytical methods'that the reducible cross-links of dehydrohydroxy-lysinonorleucine and dehydrodihydroxylysinonor-leucine were stabilized to acid hydrolysis by reduction

1975

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PULMONARY COLLAGEN

Table 3. Compositions of 'collagenase-soluble' and 'elastase-insoluble' collagen fractions preparedfrom 3-year-old cattle

Results are expressed as residues/1000 amino acid residues.

'Collagenase-soluble'

Pleura ParenchymaHypAspThrSerGluProGlyAlaValMetIleLeuTyrPheHylHisLysArgTotalCarbohydrate (g/100g)

117.545.215.632.665.0114.5325.0106.527.45.9

13.824.25.3

12.77.75.4

25.247.6

1000.11.30

95.056.521.043.786.4

107.0307.0100.017.16.3

11.733.09.3

16.18.76.5

27.647.4

1000.32.52

'Elastase-insoluble'

Pleura Parenchyma113.0 120.045.9 47.715.2 17.624.6 34.174.3 73.0115.0 99.0331.0 333.4110.0 93.524.4 22.46.5 7.114.4 15.824.6 28.34.5 6.413.6 15.88.7 7.47.0 6.8

24.5 25.443.3 45.3

1000.5 999.00.94 2.21

Table 4. Contents ofreducible cross-links in pulmonary collagen fractions and intact collagens isolatedfrom various tissues ofanimals ofdifferent ages

Pulmonary collagen fractions ('elastase-insoluble' and 'collagenase-soluble') were prepared from reduced salt-extracteddefatted pleura and parenchyma of bovine lungs. Results for parenchyma are in parentheses. Intact insoluble collagenswere isolated from bones and tendons. Concentrations of dihydroxylysinonorleucine and hydroxylysinonorleucine areexpressed as residues/300000g collagen.

'Elastase-insoluble' 'Collagenase-soluble'

Tissue sourceBovine pleural and parenchymal

fractions prepared fromanimals aged:

1 week3 years16 years

Intact insoluble collagens isolatedfrom:

1-day-old chick bones3-year-old cow bone1-week-old calf tendon3-month-old rat tendon

Dihydroxylysinonor- Hydroxylysinonor- Dihydroxylysinonor- Hydroxylysinonor-leucine leucine leucine leucine

0.19 (0.38)0.07 (0.05)

<0.05 (<0.05)

1.850.440.080.05

0.15 (0.29)0.13 (0.10)0.10 (0.09)

0.29 (0.36)<0.05 (<0.05)<0.05 (<0.05)

0.21 (0.24)0.19 (0.16)0.14 (0.15)

0.300.210.340.41

of the salt-extracted defatted pleural and parenchymalpowders with KB3H4 before digestion with enzymes.Figs. l(c) and l(d) show the distribution of 3H afterthe preliminary separation on a resin column of acidhydrolysates of 'elastase-insoluble' and 'collagenase-soluble' pleural fractions prepared from a 3-year-oldanimal. Also included in Fig. 1 are the radioactivityVol. 145

patterns given by comparable pleural fractions pre-pared from a 1-week-old animal (Figs. la and lb)and from a 16-year-old animal (Figs. le and lf).The main radioactive components at peaks 3 (di-hydroxylysinonorleucine) and 4 (hydroxylysinonor-leucine) seen in the traces of both fractions from the1-week-old animal decreased with increasing age of

293

G. FRANCIS AND J. THOMAS

6

4-

2

0.

(c)(1)(2) (3) (4) (5) (6) (7) (8)

o~~~~~~ II

5 25 35 45 55

(b)

I'

4r

(3) (4) (5) (6) (7) (8)3

2

(2) 3) 4i (5) (6) (7) (8)

s 25 35 45 55

55

(f)

() (2) (3) (4) (5) (6) (7) (8)

25 35 45 55 I5 25 35 45 55

Fraction no.

Fig. 1. Distribution of 3H in acid hydrolysates ofreduced collagen fractions from bovine pleura of animals ofdifferent ages

Salt-extracted defatted pleural powders from bovine lungs of 1-week-old, 3-year-old and 16-year-old animals were reducedwith KB3H4, the 'elastase-insoluble' and 'collagenase-soluble' collagen fractions prepared, hydrolysed with acid andseparated on a sulphonated polystyrene column. (a) 'Elastase-insoluble' and (b) 'collagenase-soluble' fractions from 1-week-old animal. (c) 'Elastase-insoluble' and (d) 'collagenase-soluble' fractions from 3-year-old animal. (e) 'Elastase-insoluble'and (f) 'collagenase-soluble' fractions from 16-year-old animal. The positions of amino acids are (I) tyrosine, (2) phenyl-alanine, (3) dihydroxylysinonorleucine, (4) hydroxylysinonorleucine, (5) hydroxylysine, (6) lysinonorleucine, (7) 'FractionC' (histidinohydroxymerodesmosine), (8) lysine.

1975

294

6

a

0

C)Cu._o

0IX64e

iI

(I1:

I I

. --A-&-A-

PULMONARY COLLAGEN

21

(b)

300~~:; 9

() (2) (3) (4) (5) (6) (7) (8) (1)(2) (3) (4) (5) (6) (7) (8)

11 , ,~,JN I

0

> I5 25 35 45 55 15 25 35 45 55

14-0

(c) 4 (d)

x(i) (2) (3) (4) (5) (6) (7) ( 3) (i)n(2) (3) (4) ( s) (6) (7) (8)

I0 (

3-

6-

2-

4-

is 25 35 45 55 IS 25 35 45 55Fraction no.

Fig. 2. Distribution of3Hin acidhydrolysates ofreduced intact collagensfrom bone andtendonThe insoluble collagens from decalcified defatted bones from chicks (1 day old) and cattle (3 years old), and from salt-extrac-ted defatted tendons from calf (1 week old) and rat (3 months old) were reduced with KB3H4, hydrolysed with acid andseparated on a sulphonated polystyrene column. (a) Chick bone; (b) bovine bone; (c) rat tendon; (d) bovine tendon. Posi-.tions of amino acids are (1) tyrosine, (2) phenylalanine, (3) dihydroxylysinonorleucine, (4) hydroxylysinonorleucine, (5)hydroxylysine, (6) lysinonorleucine, (7) 'Fraction C' (histidinohydroxymerodesmosine), (8) lysine.

295

the animal, showing that the concentrations of thesereducible cross-links decreased on aging. A similardecrease was observed in the collagen fractions iso-lated from the parenchyma of the same animals.

Vol. 145

Measurements of the reducible cross-links by usingninhydrin (see Table 4) established that the contentsin both collagen fractions prepared from pleura andparenchyma decreased with increasing age of the

G. FRANCIS AND J. THOMAS

animals. The total concentration of both cross-linksin the fractions was about 0.5 residue/300000g in a1-week-old animal, 0.2 residue/300000g in a 3-year-old animal and 0.1 residue/300000g in a 16-year-oldanimal. The concentration of dehydrohydroxylysino-norleucine was less affected by aging than that ofdehydrodihydroxylysinonorleucine, the latter beinghardly detectable in old animals. No detectableamounts of hydroxylysinonorleucine or dihydroxy-lysinonorleucine occurred at any age in pleural orparenchymal collagen fractions which had not beenreduced with KBH4. Small amounts of lysinonorleu-cine were observed in the fractions, but the concen-tration did not exceed 0.05 residue/300000g in anyfraction.

Contents of reducible cross-links in intact collagensisolatedfrom bone and tendon

Fig. 2 shows the distribution of 3H after separationon a resin column of acid hydrolysates of insolubleKB3H4-reduced intact collagens isolated from dif-ferent types of bone and tendon. All the collagensexamined contained the radioactive components atpeaks 3 and 4 and therefore contained both aldiminecross-links. However, there was a difference in therelative proportions of the components dependingon the type of collagen. Thus component 3 (di-hydroxylysinonorleucine) was the main componentin bone collagens isolated from chicks (Fig. 2a) andcattle (Fig. 2b), and component 4 (hydroxylysino-norleucine) was the main component in tendon col-lagens isolated from rat (Fig. 2c) and calf (Fig. 2d).Component 7 seen in the radioactive trace of rat tail-tendon collagen was first observed by Bailey et al.(1970) and was called 'Fraction C', but was lateridentified as histidinohydroxymerodesmosine byTanzer et al. (1973).The concentrations of the reducible cross-links

measured with ninhydrin in these collagens are in-cluded in Table 4. Apart from chick bone collagen(total of 2 residues/300000g) the other tissue colla-gens had total concentrations of about 0.5 residue/300000g. No lysinonorleucine or other elastin cross-linking compounds were observed in any collagensample.

In order to test whether any reduction in vivo ofthe aldimine bonds of the reducible cross-links hadoccurred in these collagens, attempts were made tomeasure the concentrations of dihydroxylysinonor-leucine and hydroxylysinonorleucine in acid hydro-lysates of collagens that had not been reduced withKBH4. Neither compound could be detected.The concentrations of dehydrohydroxylysino-

norleucine and dehydrodihydroxylysinonorleucine(measured in reduced forms with ninhydrin) in the'collagenase-soluble' and 'elastase-insoluble' collagen

fractions prepared from chick bone and 1-week-oldcalf tendon were very similar to the concentrations inthe intact collagens of both tissues. The 'collagenase-soluble' and 'elastase-insoluble' fractions contained80-85% ofthe total insoluble collagen initially presentin each tissue. This recovery of collagen was similarto that obtained for the same fractions isolated frompulmonary tissues.

Occurrence of e-(y-glutamyl)lysine and e-(y-glutamyl)-hydroxylysine cross-links in the bovine pleural'elastase-insoluble' collagen fraction

After cyanoethylation of 'elastase-insoluble' col-lagen, followed by acid hydrolysis, variable amountsof lysine and hydroxylysine were detected, but theamounts ofeach did not exceed 0.15 residue/300000gof collagen. In view of these small amounts and thepossibility that not all the free e-amino groups oflysine and hydroxylysine had reacted with acrylo-nitrile the presence and importance of e-(y-glutamyl)-lysine and e-(y-glutamyl)hydroxylysine cross-links incollagen is doubtful.

Discussion

Methods were examined for isolating collagen in apure form from the salt-extracted defatted visceralpleural and parenchymal regions of the lungs of 3-year-old cattle. The collagen content of both tissueswas about 70% (w/w), the remaining material beingidentified as elastin and structural glycoproteins. Thegreat insolubility of these three classes of proteinspresented difficulties when trying to isolate pure col-lagen from pulmonary tissues. The methods used werebased on the selective solubilization of either thecollagen or of elastin and structural glycoproteins.Methods involving treatments with reagents whichwould not be expected to break covalent bonds wereunsuccessful. Other methods involved digestion withproteinases of different specificities, and of the en-zymes studied collagenase and pancreatic elastaseproduced the purest forms of collagen. Digestion ofpulmonary tissues with collagenase solubilized about87% (w/w) of the total collagen, whereas digestionwith pancreatic elastase had a limited solubilizingeffect on collagen and about 87% ofthe total collagenremained insoluble. The 'collagenase-soluble' and'elastase-insoluble' materials were substantially freefrom elastin and structural glycoproteins, and chem-ical analyses showed them to be collagenous in nature.The amino acid and carbohydrate compositions ofthese collagen fractions were more similar to those ofthe interstitial collagens found in bone, skin andtendon (see Eastoe, 1967) than of the basementmembrane collagens found in glomeruli (Kefalides,

1975

296

PULMONARY COLLAGEN 297

1967). They possessed lower hydroxyproline, hyd-roxylysine and carbohydrate contents than the base-ment membrane collagens and the pleural 'elastase-insoluble' fraction was shown to contain bothgalactose and glucosylgalactose units each linked byan O-glycosidic bond to the hydroxyl group of ahydroxylysine residue, unlike the basement mem-brane collagens in which the carbohydrate occursalmost exclusively in the form of disaccharide units.Also, unlike basement membrane collagens, thepulmonary collagens contained no half-cystine.The pulmonary collagen fractions were shown to

contain the reducible cross-links dehydrohydroxyl-lysinonorleucine and dehydrodihydroxylysinonor-leucine. When expressed as residues of cross-link/300000g of collagen the values in young cattle were0.5, in mature cattle 0.2 and in old cattle 0.1. Thevalues for bone collagens in 1-day-old chicks was2 residues and in adult cattle 0.7 residue/300000g ofcollagen. The comparable value for calf tendon and3-month-old rat tendon was about 0.4. Robins et al.(1973) have suggested that the above reducible cross-links are the most recently synthesized cross-links incollagen and during maturation of animals are con-verted into more stable ones of unknown identitywithout an increase in the total. One possibility is thatthe aldimine bonds are reduced in vivo. This isknown to occur during the maturation of elastinwhere the aldimine cross-links dehydrolysinonor-leucine and dehydromerodesmosine are convertedinto lysinonorleucine and merodesmosine. However,in the present work there was no evidence that anyin vivo reduction of aldimine cross-links occurred incollagen isolated from various mammalian tissuesources.Tanzer & Mechanic (1970) have reported that

reduced preparations of calf skin collagen containedlysinonorleucine. In the present study trace amountsof this compound together with traces of desmosine,isodesmosine and merodesmosine were detected inthe pulmonary collagen fractions. It has been estab-lished that all these compounds are cross-linkers inelastin (Francis et al., 1973) and since pulmonarytissues contain elastin it was concluded that theirpresence in pulmonary collagen fractions arose fromcontamination with elastin.Another type of lysine-derived cross-link, e-(y-

glutamyl)lysine, has been shown to occur in fibrinduring blood clotting (Pisano et al., 1969) and in hairmedulla protein (Harding & Rogers, 1971). Thiscross-link and the analogous e-(y-glutamyl)hyd-roxylysine were detected in such small amounts inpleural collagen that they were regarded as artifactswithout real significance in the cross-linked collagenstructure.

The work was supported by the Science Research Coun-cil and the Royal Society. G. F. thanks the Medical Re-

search Council. We are grateful to Dr. A. J. Bailey for puresamples of hydroxylysinonorleucine and dihydroxylysino-norleucine.

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