Gut, 1969, 10, 484-487

Polymeric collagen isolated from the humanintestinal submucosa

F. S. STEVEN, D. S. JACKSON, J. D. SCHOFIELD, AND J. B. L. BARD

From the Department of Medical Biochemistry, Lapworth Laboratories, and the Biophysics Unit,Rheumatism Research Centre, University of Manchester, Manchester

The intestinal submucosal layer has a special interestin view of its commercial use in sausage casings andalso due to its importance in any study of normal andpathological changes in the human intestine.Although the submucosal layer has long been knownto be a collageneous structure, no detailed reporthas been published for the isolation of highly puresubmucosal collagen together with its chemicalanalysis and electron-microscopic appearance.

Earlier work from this laboratory has beenconcerned with the isolation and characterization ofpure polymeric collagens from tendon and skin(Steven, 1964; Steven and Jackson, 1967), cornea(Freeman, Steven, and Jackson, 1968), intervertebraldisc (Steven, Broady, and Jackson, 1968), and arti-cular and intercostal cartilages (Steven, Broady, andJackson, 1969). In each of these preparations it wasnecessary to pretreat the homogenized tissue withcrude bacterial oc-amylase or ethylenediaminetetra-acetate (EDTA) in order to release the collagenfibrils from the tissue matrix in which they wereembedded.

In the present study the polymeric collagenfibrils of the intestinal submucosa could be releasedby treatment with dilute acetic acid alone. Theamino-acid composition of these polymeric collagenfibrils indicated that they consisted of at least 95%pure collagen. The purity of the polymeric collagenwas further improved after treating the tissue before-hand with crude bacterial oc-amylase or with EDTA.

MATERIALS AND METHODS

PREPARATION OF TISSUE Human intestines were removedas soon as possible at necropsy from subjects with noknown intestinal abnormality. The tissues were carefulllywashed with water and the submucosal layer was scrapedclean of the mucosa and muscularis layers. Segments of thesubmucosal layer from both the small and large intestinewere removed and separately frozen in liquid nitrogen.The tissues were crushed in a stainless steel hammer mill(Steven, 1967; Walser and Bodenloss, 1954) and exhaus-tively washed in 0-5 M NaCl by centrifugation until the

supernatant was clear and contained no solubilizedprotein.

ACETIC ACID EXTRACTION Part of the homogenized tissuewas suspended in 5 1. 0-2 M acetic acid and allowed toequilibrate to pH 3.2 to 3-5 over a period of one hour.The tissue was then homogenized in an Utraturraxhomogenizer for 10 seconds, followed by constantstirring at 40 for 18 hours. The supernatant fluid contain-ing polymeric collagen fibrils was removed by centrifuga-tion at 300g for20min at 4°C and the fibrils were collectedby raising the pH to 5 to 6 with M.NaOH and stirringwith a spatula (Steven, 1964; Steven and Jackson, 1967).Polymeric collagen fibrils were purified from non-collageneous proteins by redispersing in acetic acid fourtimes followed by precipitation of the fibrils by theaddition of saturated NaCI to give a final concentrationof 3 to 4% w/v.

GELATIN FORMATION Acetic-acid-extracted polymericcollagen fibrils were autoclaved at pH 7.0 for six hr at15 lb/in.2; the insoluble residue contained 94% of theinitial collagen and the soluble fraction 6% respectively.The soluble fraction was purified as a gelatin afterprecipitation of non-collagenous impurities with 5 %trichloracetic acid and precipitation of the gelatin with10 volumes of acetone (Jackson, 1957). The insolubleresidue was autoclaved in water for a further six hr at30 lb/in.2 but failed to yield any further soluble gelatinfraction.

CRUDE a:-AMYLASE TREATMENT Part of the tissue homo-genate was treated with crude a-amylase, 1% w/w, aspreviously described for tendon and skin (Steven, 1964;Steven and Jackson, 1967), and the polymeric collagenfibrils were collected and purified as above, all stepsbeing carried out at 4°C.

EDTA TREATMENT Part of the tissue homogenate wastreated with 4% w/v EDTApH 7.5 (Steven, 1967) and thepolymeric collagen fibrils were isolated and purified asabove.

AMINO ACID ANALYSIS The collagen and gelatin prep-arations were subjected to 24- hr hydrolysis under nitrogenin 6N HCI and amino acid analysis was carried out on the

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Polymeric collagen isolatedfrom the human intestinal submucosa

21-hour Technicon sodium buffer system as previouslydescribed (Steven, 1967; Steven and Jackson, 1967).

ELECTRON MICROSCOPY Samples of submucosal poly-meric collagen fibrils dispersed in 0-1 M acetic acid wereplaced on carbon-collodion-coated grids and negativelystained with 1 % w/v sodium phosphotungstate pH 7.0.The grids were examined in the AEI-EM-6B electron-mnicroscope.

RESULTS AND DISCUSSION

YIELD OF POLYMERIC COLLAGEN Virtually all thecollagen of the submucosal tissue was capable ofdispersion into 0.1 M acetic acid as polymericcollagen fibrils, and less than 10% remained in theinsoluble residue after two extractions with aceticacid. The yield was not affected by pretreatment ofthe tissue with either crude a-amylase or by EDTA.The ease with which dilute acetic acid disperses

the polymeric collagen fibrils of the intestinalsubmucosa is in marked contrast to the difficultiesencountered with other connective tissues. Skin andtendon require a pretreatment with crude bacterialoc-amylase (Steven, 1964; Steven and Jackson, 1967)or EDTA (Steven, 1967) before the polymericcollagen fibrils can be dispersed in acetic acid. Thesepretreatments do not release pure polymericcollagen from human cartilage unless it is furthertreated with a proteolytic enzyme such as trypsin(Steven et al, 1968 and 1969). Bone decalcifiedwith EDTA and treated with crude oc-amylasefollowed by trypsin cannot be dispersed in acetic

acid to yield pure polymeric collagen fibrils.It is suggested that the submucosal polymericcollagen fibrils have very little interaction with othernon-collageneous components of this tissue andthat they represent the least complex organizationof polymeric collagen fibrils at this tissue levelso far studied.

DEGREE OF POLYMERIZATION OF POLYMERIC COLLAGENProlonged autoclaving of collagen is known toproduce peptide bond cleavage with the formation ofa soluble polydisperse polypeptide fraction usuallyreferred to as gelatin. Human skin and tendonpolymeric collagen is completely solubilized afterone hour's autoclaving. The marked insolubilityof submucosal polymeric collagen after 12 hours'autoclaving indicates a very high degree of inter-molecular cross-linking or polymerization withinthe polymeric collagen fibrils. These crosslink-ages are stable and prevent the solubilization of poly-peptide fragments resulting from peptide bondcleavage during autoclaving.

AMINO ACID COMPOSITION OF POLYMERIC COLLAGENThe analyses presented in Table I indicate that boththe crude oc-amylase and EDTA-prepared polymericcollagen fibrils were highly purified. The polymericcollagen fibrils extracted with acetic acid alonecontained some 6 to 7% impurity calculated on thecontent of hydroxyproline and proline as well asglycine residues. This impurity could be removedfrom the polymeric collagen by autoclaving and

TABLE IAMINO ACID COMPOSITIONS OF POLYMERIC COLLAGENS AND DERIVED GELATIN OBTAINED FROM HUMAN INTESTINAL

SUBMUCOSA1Acetic-acid ExtractedPolymeric Collagen

81-354.219232-380.2

105-3313.7117-234.41.9

16-031.54.51445.32-7

30-2650

49-6999.9

Derived Gelatin(Gel-])

95 154-116336-174-2115-7339.7105 5234

Trace12-125-02.51117.52-0

28-44.7

46-61,000 0

Polymeric Collagen

a-Amylase EDTA

97.151*418.435-877.4

110-1315-9111128.12-114427-14-214-37-21-1

26-87-2

50.2

999.9

95.452416-439 077-8112-6315-4118-427-2

Trace12-026.72-7

12-85.7

Trace29-36-0

50.2

1,000-0

'Results have been expressed as residues per 1,000 total residues after allowing for hydrolytic losses (Steven and Jackson, 1967).

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Preparation

HydroxyprolineAspartic acidThreonineSerineGlutamic acidProlineGlycineAlanineValineMethionineIsoleucineLeucineTyrosinePhenylalanineHydroxylysineOrnithineLysineHistidineArginineTotal

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F. S. Steven, D. S. Jackson, J. D. Schofield, and J. B. L. Bard

A

L.E

A

I,.

B

C

FIG. 1. Intestinal submucosal polymeric collagen fibrils negativelv contrasted with sodium phospho-tungstate (pH 7.0) x 50,000, showing fibrils swollen but still maintaining 640 A4 periodicity (A)andswollen stocking-like structure (B) in addition to unswollen native type collagen fibrils (C).

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Polymeric collagen isolatedfrom the human intestinal submucosa 487

purifying the derived gelatin (6% of the total poly-meric collagen) by trichloracetic acid and acetoneprecipitation (see figures for gel-1, Table 1).The hydroxylysine content ofintestinal submucosal

polymeric collagen is lower than tendon and cartilagepolymeric collagen (Steven et al, 1969). The sum oflysine plus hydroxylysine polymericcollagen obtainedfrom these tissues is approximately constant as wouldbe expected from the fact that hydroxylysine isderivedfrom lysine in protocollagen (Kivirikko andProckop,1967; Prockop and Kivirikko, 1967).

ELECTRON MICROSCOPIC EXAMINATION OF POLYMERICCOLLAGEN Examination of submucosal polymericcollagen fibrils dispersed in 0 1 M acetic acid demon-strated the typical 640A striated fibrils (A, Fig. 1) aswell as fibrils which were partially unravelled(B, Fig. 1). The protofibrils which make up thefibril can be seen to be organized in the form of a'stocking' type of structure, with some evidence ofsuperhelical organization. Stirtz (1967) has alsopublished evidence suggesting a superhelical organ-ization of protofibrils within collagen fibrils. Theswollen fibrils (B in Fig. 1) appear unable to becompletely unravelled. This suggests that a limitednumber of interprotofibrillar bonds may be presentand that these place a restriction on the dispersal ofthe protofibrils in dilute acetic acid.

It may be concluded that the human submucosacontains polymeric collagen fibrils which are veryloosely held in the interfibrillar matrix and that diluteacetic acid readily extracts at least 90% of the poly-meric collagen in the form of dispersed fibrils. Thepolymeric collagen fibrils are composed of a largenumber of superhelically organized protofibrils,whilst at the molecular level there must be a highdegree of intermolecular cross-linking which isstable to autoclaving in marked contrast to otherpolymeric collagens so far studied.

SUMMARY

Treatment of human intestinal submucosa withdilute acetic acid allowed the dispersal of polymericcollagen fibrils which were purified by salt precipita-tion. These fibrils were 97% pure collagen. Treat-ing the tissue beforehand with crude bacterialoc-amylase or EDTA resulted in the dispersion ofpure polymeric collagen fibrils. The amino acidcomposition and the electron microscopic appear-ance of intestinal submucosa polymeric collagenfibrils are reported.

We wish to thank Dr A. Jones for provision of theintestines and Miss K. Broady for her excellent work onthe Technicon AutoAnalyzer system.

J.D.S. and J.B.L.B. wish to thank the S.R.C. forresearch studentships held during the course of this study.

REFERENCES

Freeman, I. L., Steven, F. S., and Jackson, D S. (1968). Isolationand amino acid composition of bovine corneal polymericcollagens. Biochim. biophys. Acta (Amst.), 154, 252-254.

Jackson, D. S. (1957). Connective tissue growth stimulated bycarrageenin. 1. The formation and removal of collagen.Biochem. J., 65, 277-284.

Kivirikko, K. I., and Prockop, D. J. (1967). Enzymatic hydroxylationof proline and lysine in protocollagen. Proc. nat. Acad. Sci.(Wash.), 57, 782-789.

Prockop, D. J., and Kivirikko, K. I. (1967). Relationship of hydroxy-proline excretion in urine to collagen metabolism. Ann. intern.Med., 66, 1243-1266.

Steven, F. S. (1964). The Nishihara technique for the solubilization ofcollagen. Ann. rheum. Dis., 23, 300-301.

(1967). The effect of chelating agents on collagen interfibrillarmatrix interactions in connective tissue. Biochim. biophys.Acta (Amst.), 140, 522-528.

, Broady, K., and Jackson, D. S. (1968'. Ibid., 160, 435-446.- ,-,- (1969). Ibid., 175, 225-227.

, and Jackson, D. S. (1967). Purification and amino acid com-position of monomeric and polymeric collagens. Biochem. J.,104, 534-536.

Stirtz, T. (1967). Leder, 18, 193-204.Walser, M., and Bodenlos, L. J. (1954). Composition of skin as com-

pared with muscle. Amer. J. Physiol., 178, 91-96.

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