Dermatan sulfate-protein: Isolation from and interaction with collagen

ARCHIVES

OF

BIOCHEMISTRY

AND

Dermatan

128, 567-578 (1968)

BIOPHYSICS

Sulfate’-Protein: Interaction

l-3. P. TOOLE2 Department

of Biochemistry, Received

March

Isolation

with

from

and

Collagen

D. A. LOWTHER

AND

Monash

University,

19, 1968; accepted

Clayton,

Victoria,

Australia

July 26, 1968

A dermatan sulfate-protein compIex cont.aining approximateIy 50y0 noncoIIagenous protein and less than 47, of collagen, has been ext,racted in 6 M urea at 60” from the fibrous residue of heart valves, and separated from the solubilixed collagen by precipitation with ethanolic KCNS. The preparation was fractionated into two components by cesium chloride gradient centrifugation. The major component contained no detectable collagen and no free protein was separated. The dermatan sulfateprotein has markedly different physical properties to hyaluronateand chondroitin sulfate-proteins but., when mixed with tropocollagen at physioIogica1 ionic strength and pH, it gives rise to an immediate precipitate containing “native-type” collagen fibrils. Similar dermatan sulfate-proteins that also precipitate tropocollagen have been prepared from bovine skin and tendons. It is therefore proposed that the primarv bioloaical role of dermatan sulfate-protein may be in the formation and orientation of collagen fibers.

Chondroitin-4-sulfate, chondroitin-6-sulfate, keratan sulfate, and hyaluronate have been extracted with water or neut’ral salt solution from many tissues by homogemization and found to be associated with noncollagenous protein (1,2). However, it has been reported that the dermatan sulfate (DS) of skin and heart valves cannot be extracted with various salts and hydrogen bond-breaking solvents, but appears to be firmly bound to the collagen fibers of these tissues (3-5). In previous papers by the authors, it was claimed that a Iarge proportion of t,his DS could be extracted by extensive homogenization in cold 6 M urea (4, 6). In subsequent experiments, however, the amounts obtained by this method were found to be very variable and occasionally less than 1% could be extracbed. In this study most of the DS of

these tissues is found to be extracted on gelat’inization of the collagen fibers in 6 M urea at 60”. Preliminary work on DS obtained from prot’eolytic digests of skin suggests that this polysaccharide exists as a complex with peptide material in situ (3, 7-9) and t’his paper describes t,he isolation and purification of DS-protein complexes containing approximately 40 % polysaccharide and 60 % protein from the hot urea extracts of heart valves, skin, and tendon. These preparations react with tropocollagen at physiological pH and ionic st,rength t,o form immediately a precipitate that contains “native-type” collagen fibrils and continued incubation at 37” yields a gel having a fibrous appearance.

1 Chondroitin sulfate-B. 2 This investigation fulfills part of the requirements for the degree of Doctor of Philosophy, Monash Universit,y. Present address: Developmental Biology Laboratory, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts 02114.

Collagenous residues were prepared heart valves, skin, and pooled patellar lar tendons from the knee joint after the soluble material of t,he tissues by exhaustive homogenizations in water I M NaCI at O-5” (4, 5). Ethantrlyzed cellulose was prepared 567

Copyright

@ 1968 by Academic Press Inc.

MATERIALS

AND

JIETHODS from bovine and capsuextraction of repeated and followed by from cotton

568

TOOLE AND LOWTHER

wool by boiling in an ethanol-HCI mixture and washing thoroughly (10). 6 M urea solutions were passed through a mixed-bed resin prior to use. Partially purified papain was obtained from Mann Research Laboratories, bovine testicular hyaluronidase from Evans Medical Ltd. The reference compound, DS, was prepared from proteoIytic digests of skin as described previously (4) but in addition was treated exhaustively with testicular hyaluronidase to remove possible contamination with hyaluronate or chondroitin sulfate. Chondroitin sulfate-protein from bovine nasal cartilage was prepared from water extracts of the tissue by quaternary ammonium salt precipitation (11). Hyaluronateand chondroitin-sulfate proteins from bovine heart valves were isolated from water extracts by density gradient centrifugation in CSCI (12, 13). DS was measured in crude fractions derived from the tissue residues by digestion of the fractions with papain followed by dialysis, concentration, and assay with Rivanol using the reference DS as standard (14). Total hexosamine (15) and galactosamine (16) were measured after hydrolysis of samples in 4 M HCl at 100’ for 16 hr. Glucosamine was calculated by subtracting galactosamine content from total hexosamine content. Uranic acid was measured by the methods of Bitter and Muir (17) and of Dische (18) using glucuronolactone as a standard. When the values obtained in the two procedures were similar, the uranic acid component was assumed to be glucuronic acid. However, in the case of the uranic acid component of DS, i.e., iduronic acid, the value obtained by the Dische procedure is approximately 50% of that obt,ained by the Bitter and Muir method when glucuronolactone is used as standard in both methods (see 17). Sulfate was determined by the method of Antonopoulos (19), protein by the met.hod of Lowry et al. (20), and collagen by calculation from hydroxyproline analyses (21) using a factor of 7.5. Amino acid analyses were performed on a Beckman Spinco Automatic analyzer after hydrolysis of samples in uacuo in 6 M HCI at 105” for 18 hr. Sensitivity to hydrolysis by testicular hyaluronidase was estimated as described before (5). C&l density gradient centrifugation was carried out in the SW 39 rotor of the Spinco Model L2 preparative ultracentrifuge at 35,000 rpm for 16-24 hr (12). Solid CsCl was added to the proteinpolysaccharide solution at 4O to give the required density (22). Fractions were obtained with a Beckman tube-sIicer or by colIecting drops from a hole placed in the bottom of the tube. The density of each fraction was measured using a constriction pipette. Uranic acid analyses (17) were carried out directly on each fraction and both uranic acid

and protein were assayed after dialysis against running tap wat,er, 1 M NaCI, running tap water and then distilled water. Column electrophoresis was performed in 0.05 M acetate buffer, pH 5.0, containing 6 M urea on ethanolyzed cellulose or in 0.025 M ph0sphat.e buffer, pH 7.5, on untreat,ed ceIIulose. Both types of experiment were run at 15-20 mA for 16 hr on a Porath column supplied by LKB Produkter. The separated products were then eluted and analyzed for uranic acid and protein. Free boundary electrophoresis was carried out in 0.1 I barbitone buffer pH 7.6 in a MSE Model D apparatus. Electron micrographs of collagen fibrils were obtained using a Hitachi HU IlA microscope. Specimens were prepared by depositing dilute suspensions of fibrils on copper mesh grids, drying, and staining with neutral 1.5y0 phosphotungstic acid at 4”. EXPERIMENTAL

AND

RESULTS

ISOLATION AND FRACTIONATION OF DS-PROTEIN

Extraction of DS from tissue residues in 6 M urea. The effect of temperature and time of extraction on the release of DS from heart valve residue by 6 M urea solution has been investigated. At CklO” the extraction was not reproducible despite thorough and repeated dispersion of the residues in a high-speed blade homogenizer and the amounts extracted were in the range 140 % of the total DS in the residues. When the temperature was raised to 40 or 50”, the additional amount of DS extracted was still less than 10 %. However, at 60” a large proportion of the DS was reproducibly extracted. The actual amount that dissolved depended on time of extraction and stirring. As shown in Table I, 86-94% of the DS could be extracted in 16 hr without stirring, whereas only 25% went into solution in 2 hr. The yield after 2 hr could be increased to 66-86 % by stirring the suspension vigorously throughout the extraction. The latter method was used routinely in the preparation of DS-protein described below. Separation of DS-protein and gelatin in the 6 M urea extracts. DS-protein was selectively precipitated from the urea extracts by the addition of sat,urated ethanolic KCNS (2.3 vol). Precipitation was carried out by gradually adding the ethanolic KCNS to the extracts with constant stirring at 4”. The

DERMATAN

TABLE OF DS-PROTEIN

EXTRACTION

569

SULFATE-PROTEIN

FROM HEART

I

VALVE

AND SKIN

BY 6 M UREA

RESIDUES

AT 60”

Tissue Heart valve

Method Time Batch

Vigorous

of extraction of extraction

No stirring

stirring 2 hr

2 hr I

II

66

86

no.

y0 DS extracted

III 70

OF ETHANOLIC

Ratio of saturated extract Recovery Protein

KCNS

ethanolic

NEEDED

KCNS

IV

v

VI

67

25

94

86

of DS (%) to DS ratio

in precipitate

AXD PURIFICATION

94

0

0

99

-

-

1.6

3:l

2.3:1

DS extracted in 6 M urea at 60” (7’ of total in residue) Collagen:DS ratio (w/w) in urea extracts DS in DS-protein solution (70 of total in extracts) Collagen:DS ratio (w/w) in DS-protein solution (1Batches

I-IV

were extracted

II

EXTRACTS

4:l

6:l

99

110

110

2.0

3.2

4.0

WITH

III

IV

v

ETHANOLIC

Skin

KCNS

Tendon

66

86

70

67

94

88

-

15

-

50

30

60

530

44

98

73

93

75

99

100

-

0.11

0.12

0.26

0.39

0.25

0.18

0.09

in urea for 2 hr, batch V for 16 hr.

was left to stand for at least 1 hr at 4”, then centrifuged at 5000g for 30 min. The precipitate was washed once with saturated ethanolic KCNS: water (7: 3 v/v) and finally three times with 95% ethanol. The DS-protein was redissolved by stirring in water and then dialyzed against water overnight at 4”. In the precipitation procedure described above, the composition of the final solution was found to be critical (see Table II). Addition of 2 vol of ethanolic KCNS yielded

suspension

88

III BY PRECIPITATION

Heart valve (batch no.)& I

89

FROM HOT UREA

2:l

OF DS-PROTEIN

VIII

II

1:l

TABLE RECOVERY

16 hr

VII

TO PRECIPITATE DS-PROTEIN OF HEART VALVES

to

No stirring

16 hr

IV

TABLE AMOUNT

Skin

no precipitate while addition of 3-6 vol led to the precipitation of extraneous protein. In addition, if the ethanolic KCNS was added too rapidly or if mixing during this step was inefficient, considerable precipitation of collagen occurred and the DS-protein was difficult to dissolve in water. In such cases, however, the precipitate could be redispersed in 6 Y urea at 60” for 5 min and then precipitation repeated. The final solution of DS-protein was clarified by centrifugation at approximately 120,OOOg (Spine0

570

TOOLE AND LOWTHER TABLE COMPOSITION

Batch no.

Percentage composition I II Molar ratiosb 1 II

IV

OF HEART

Total hexosamine

Galactosamine

12.0 11.8

10.5 10.9

1.00 1.00

VALVE

DS-PROTEIN

Uranic acidm Method A Me20d

Sulfate

Protein

Collagen

10.1 10.0

5.2 4.6

5.1 4.8

49 50

3.7 3.8

0.37 0.36

0.80 0.76

-

-

0.88 0.92

0.78 0.78

0 Uranic acid was measured by the method of Bitter and Muir (Method A) (17) and by the method of Dische (Method B) (18). Iduronic acid (the uranic acid component of DS) has been found to give approximately 80’% of the yield of color given by glucuronolactone (the standard used in the assays) in the former met,hod and approximately 40y0 in the latter (see 17). b Total hexosamine taken as 1.00. TABLE COMPARISON

OF DS-PROTEIN

V

PREPARATIONB

FROM

HEART

VALVE.

SKIN.

AND

TENDON

Tissue Heart valve (batch no.) I

Ratio protein to DS Collagen (as y0 protein) Uranic acida, ratio of Method B to Method A Test for hydrolysis by testicular hyaluronidase ($& reduction in absorbance) Optical rotation-(&+

II

III

Skin

Tendon

Reference DS (pigskin)

1.37 7.6 0.47

1.20 0.43

1.64 11.2 0.55

1.22 7.6 0.60

0.59

16

0

6

15

0

5

-53”

-

-

-

-53”

1.43 7.6 0.46

-67”

a Method A, Bitter and Muir (17) ; Method B, Dische (18). The ratio obtained for DS by Bitter and Muir was approximately 0.5. b The polysaccharide obtained by quaternary ammoniumsalt precipitation after proteolytic digestion of the DS-protein complex was used for measurement of optical rotation.

Model L rotor no. 50). The percentage of DS-protein recovered after this precipitation procedure varied from 73-100% of the total content in the extracts (Table III). The collagen (assuming hydroxyproline represents collagen only) to DS ratio was measured in the original urea extracts and the isolated DS-protein and found to be reduced by a factor of lOCk3000 by the ethanolic KCNS precipitation (see Table III). From 5 to 40% of the DS-protein obtained after redispersion in water was sedimented by prolonged centrifugation at 120,OOOg and contained somewhat higher proportions of total and collagenous protein than that remaining in the supernatant fluid. However,

the amount sedimenting depended greatly on the time and mode of storage of the preparation prior to centrifugation. Even when the preparation was centrifuged exhaustively, new aggregates that could be sedimented at 12O,OOOg,were formed during storage in the frozen state. Consequently, these aggregates were removed prior to analysis, physical measurements, or fractionation in CsCl gradients. Analysis. The composition of two heart valve DS-protein preparations are shown in Table IV and after correcting for molar equivalents of acetyl and potassium, account for 85% of the dry weights in each case. The identification of the acid muco-

DERMATAN

Collagenous

~

571

SULFATE-PROTEIN

-residue

1 ~ ;:

of tissue

(see

Materials)

~,..t::~g~~;~o~~~~~~~~~~~~~-s

Extract !

3esidv.e

a. 2.3 vol.saturated ethanclic b. Stand, 4', l-18 hours c. Centrifuge, 5@OCg, 50 mine. d. Wash precipitate, KCNS, 3 x ethanol.

1 x ethanolic

4 Supernatant

J Precipitate a. Dissolve in b. Dialyse vs. c. Centrifuge,

water water 12O,OOOg,

5-7 hours .

r V

V

DS-protein

DS-protein

solu~tion

Chemical analysis, Physical measurements, CsCl density gradients, FIG. 1. Procedure valve,

KCNS.

skin,

sediment

etc.

for the isolation and tendon.

of DS-protein

polysaccharide component as DS was deduced from its resistance to hydrolysis by testicular hyaluronidase, it,s optical rotation, and the low color value for uranic acid by the Dische method (Table V). The small amount of hydrolysis by hyaluronidase obtained in some preparations indicates that another acid mucopolysaccharide was sometime present in small amounts (see section on C&l density-gradient centrifugation). It

from the collagenous

residues

of heart

was noted also that the skin and tendon preparations contained considerable amounts of a glucosamine-containing component that was not an acid mucopolysaccharide. The protein component of the heart valve preparations comprised approximately 50 % of the dry weight (or 59 % of t’he dry weight accounted for). The protein to acid mucopolysaccharide ratios calculated for prepara-

572

TOOLE

AND

LOWTHER

TABLE

VI

AMINO ACID ANALYSIS OF HEART VALVE DS-PROTEIN” Chondroitin sulfate-protein of nasal cartilageb

DS-protein of heart valves

LYS His Arg Asp Thr Ser Glu Pro QY Ala Val Met Is0 Leu Leu Tyr Phe OH-pro

Batch I

Batch II

46 19 51 122 56 67 139 75 90 77 50 5 37 96 23 33 14d

45 17 55 121 56 81 136 72 88 71 45 4 35 102 16 41 15d

Batch III 61 23 42 110 59 67 145 75 106 75 52 4 35 91 22 32

o Residues/1000 amino acid residues. b (1) Pal, Doganges, and Schubert (27); (2), Goh and Lowther c Gotte, Meneghelli and Castellani (23). d By separate calorimetric analysis (211. b 1

I

I

1.60

1.65

1.60

1.7c Density

(g

1.65

I

L

1.i

c.c.) /

FIG. 2. Comparison of cartilage chondroitin sulfate-protein (a) and heart valve DS-protein in CsCl density gradients (b). Initial density 1.65 g/cc. *AMPS = acid mucopolysaccharide.

tions from different sources are given in Table V and were 1.20-l .43 for heart valve DS-protein, 1.64 for skin, and 1.22 for tendon. The amino acid composition of the protein component of heart valve preparations is compared with that of chondroitin sulfate-protein from bovine nasal cartilage in Table VI. The analyses show an over-all

1

2

32 20 47 93 71 89 120 91 121 69 58 9 35 72 24 36

35 16 41 84 52 66 124 97 131 78 69 7 38 77 34 44 2d

El&in”

8 Trace 5 9 10 8 25 111 297 240 140 Trace 28 62 20 26 11

(28).

similarity, however, there are significant, differences in the contents of glutamic and aspartic acids, lysine, and glycine. It has been assumed that hydroxyproline represents collagen in this study. This amino acid is also present in elastin (23) but only in small amounts so that virtually all of the protein component of D&protein would be elastin if the latter was to account for the hydroxyproline in DS-protein. The high contents of glycine, alanine, and valine in elastin relative to DS-protein (Table VI) would preclude this possibility. Fractionation by CsCl density-gradient centrifugation. CS-proteins have been found to have a density greater than 1.8 g/cc thus allowing t’heir separation in CsCl density gradients from free protein (density less than 1.4 g/cc) (12, 13). However, when DS-protein was centrifuged in CsCl at a density of 1.65 g/cc, 85-95 % of the acid mucopolysaccharide accumulated at the top of the gradient, where the density was approximately 1.6 g/cc, and only 5-15% of the acid mucopolysaccharide sedimented to the bottom of the gradient (density of approximately 1.7

DERMATAN

SULFATE-PROTEIN

,573

0.4O’*-\ 1.39 F top

p; 1.44

1.49 f bottom

h

C

Density

(9

/

c.c.1

FIG. 3. C&l density-gradient fractionation of heart valve DS-protein. a. DS-protein preparation at initial density, 1.45 g/cc. b. Bottom fraction from a. recentrifuged at initial density, 1.55 g/cc. c. Top fraction from a. recentrifuged at initial density, 1.38 g/cc. -O--O-, DS; -O---O--, protein.

g/cc) (Fig. 2). The top fraction had a ratio of protein to acid mucopolysaccharide of 1.32, whereas the sedimented fraction had a ratio of 0.22. Measurement of the ratio of values for uranic acid by t.he Dische and Bitter and Xuir methods showed that the sediment.ed fraction was mainly contaminating CS-protein and that the less dense fraction contained the bulk of DS-protein. Centrifugation of the DS-protein at a density of 1.45 g/cc gave two fractions, one at the top of the gradient, representing 2040 % of the DS and one at the bottom of the gradient, representing 60-80 % (Fig. 3a). The top fraction was recentrifuged at 1.40 g/cc, the bottom fraction at 1.55 g/cc. In each case the DS-protein banded towards the center of the gradient (Figs. 3b and c) indicating that each fraction consisted of

molecules of similar density. However, since the subfractions within each of the two bands did not have constant protein to acid mucopolysaccharide ratios (Figs. 3b and c), it is apparent that both components exhibited further microheterogeneity. When the original preparation was examined by free boundary electrophoresis heterogeneit,y was again observed. This is indicated by the spreading of the ascending boundary shown in Fig. 4. Thus, the DS-protein preparation from heart valves cont.ains two components together with a small amount of contaminating chondroitin sulfate-protein. The major DSprotein fraction, banding at 1.55 g/cc contained 50655 % protein and 45-50 % DS by weight. The second component, banding at 1.40 g/cc, contained approximately 70% protein and 30% DS, but, varied considera-

TOOLE

AND

LOWTHER

38

FIG. 4. Free boundary electrophoresis of heart valve DS-protein in 0.11 Verona1 buffer, pH 7.5. The ascending boundarv is shown at three different times after the commencement of electrophoresis.

bly in composition from preparation to preparation. The latter component consistently included all of the hydroxyproline present in the original preparation. It appears that the different protein content of these fractions allows their separation in CsCl gradients, since the sulfate content of the DS was the same in both cases and so did not contribute to t’heir differences in density. This observation provides evidence that the protein is bound to the DS in both fractions. Furthermore, electrophoresis at 4” on a Porath column at pH 5 in urea and at pH 7.5 in the absence of urea failed to separate any free protein. PRECIPITATION OF TROPOCOLLAGEN BY D&PROTEIN

As reported previously (24), the DS-proteins prepared from extracts of heart valve, skin, and tendon by ethanolic KCNS precipitation give an immediate precipitate when mixed with tropocollagen in 0.14 M NaCl/ 0.008 M phosphate buffer, pH 7.3, at 4” or at 37” (the former temperature was normally employed to prevent spontaneous precipitation of tropocollagen (25, 26). The precipi-

t,ate formed at 4” was examined in the electron microscope and found t,o contaia collagen fibrils with banding of 550-650 A periodicity (Fig. 5). The number of fibrils observed was considerably less than that in the thick network formed when the tropocollagen is incubated at 37”. However many “native-type” fibrils were observed consistently throughout several experiments with separate batches of materials and no fibrils of this nature were seen in samples of the original solutions of DS-protein or tropocollagen. When the hyaluronate-protein or chondroitin sulfate-protein components of heart, valves were mixed with tropocollagen at 4”, no precipitate was obtained. The ratio of heart valve DS-protein t.o tropocollagen needed to give maximum precipitation was determined by turbidity measurements (Fig. 6) and found to be approximately 0.3. When precipitates obtained by mixing these proportions of reactants were separated by centrifugation at 120,OOOg for 13 hr and analyzed, they were found to contain 70% of the total DS-protein and 55% of the total collagen present’ in the

1)EI:RIATAN SULFATE-PROTEIN

FIG. 5. Collagen fibrils formed from mixing tropocollagen and I@-protein at 4”. X30,000.

original mixture. The remaining collagen in the supernatant fluid gave no precipitate when further DS-protein was added. The amount of DS-protein reacting was checked by mixing several batches of tropocollagen and DS-protein at ratios cont’aining less of the latter than needed to give maximum precipitat’ion (thus ensuring that DS-protein was limiting) and found to be consistently in the range 64-72 %. When the two components separated by CsCl densitygradient centrifugation were mixed separately with tropocollagen, only the major

fraction, \vhich banded at 1.55 g/cc (Fig. 3a and b), gave a precipitate, thus explaining why onl~ 64472% of the unfractionabed DS-protein preparation was found in the precipitates described above. Since only 55 % of the collagen appeared to react wibh DS-protein at 4”, a mixture of DS-protein and tropocollagen was incubated at 37” until precipitation of collagen was comp1et.e (as judged by t,urbidity measurements (26)). The final gel was fibrous in appearance, whereas gels obtained in the same way with tropocollagen alone, tropocollagen

576

TOOLE AND LOWTHER

DS ~protein

(mg/ml)

FIG. 6. Titration of tropocollagen with heart valve DS-protein. Tropocollagen concentration, 2900 pg/ml. The point of intersection of the two lines represents the limiting ratio of DS-protein and tropocollagen giving maximum precipitation.

FIG. 7. Gross appearance of gels obtained after incubation for 16 hr at 37”. a. Tropocollagen (2900 fig/ml) plus DS-protein (700 pg/ml). b. Tropocollagen alone (2900 ag/ml). Mixtures of tropocollagen plus heart valve hyaluronate-protein, heart valve chondroitin sulfate-protein or protein-free DS are identical in appearance to this gel.

plus heart valve chondroitin sulfate-protein, or hyaluronate-protein or tropocollagen plus protein-free DS were homogeneous and “jelly-like” (Fig. 7) (see also ref. 26). DISCUSSION

A procedure for the isolation of a DS-protein complex from the collagenous residues of bovine heart valves, skin, and tendon has been described. The method involves extraction of soluble acid mucopolysaccharideprotein complexes in water and 1 M NaCl followed by the extraction of DS from the insoluble fibrous residues in 6 M urea at 60”. The DS-protein can be isolated apparently

free from extraneous protein by fractionation with ethanolic KCNS (Fig. 1, Table III). The complex isolated from heart valves contains approximately 50 % protein and 35% DS by weight (Table IV) and has a weight average molecular weight of approximately l-2 X lo5 (29). It can be fractionated into two components containing different proportions of protein to DS by densitygradient centrifugation in CsCl (Fig. 3). This fract.ionation also results in the removal of a small amount of contaminating chondroitin sulfate-protein (Fig. 2). Since DS chains isolated after prot’eolytic digestion have a molecular weight of approximately 25,000 (30) and since free protein could not be separated from the DS-protein in the CsCl gradients or by column electrophoresis, it seems that the protein in the complex is covalently bound. The protein component contains approximately 8 % collagen (Table V) but no collagen was found in the major fraction banding at 1.55 g/cc in the CsCl gradients. It seems then that the bulk of the DS-protein is not covalently bound to collagen, but attains its close association with the collagen fibers through electrostatic or hydrogen bonding or physical entanglement. We have reported previously that amount’s of DS-protein varying from approximately l&50% of the total in the heart valves can be extracted in 1 M NaCl and a further O-40% in cold 6 M urea (5, 6). However, these preparations had considerably lower protein contents and extrapolated sedimentation coefficients than those obtained in this present study (6, 31) and consequently may represent material degraded either during preparation or by cat,abolic processes in the tissue itself. The physical properties of chondrotin sulfate-proteins

and hyaluronate-proteins

from

several tissues indicate that these macromolecules interact in solution to form networks (32). These networks in turn can become entangled with fine collagen fibrils to form highly hydrated gels (33,34). Chondroitin sulfate- and hyaluronate-prot’eins are readily ext,racted after physical dispersion of the particular tissue (1, 2, 4, 5) and it thus seems that these components are mainly

involved

in forming

a gel matrix

DERMATAN

SULFATE-PROTEIN

between fibers that contributes to the mechanical properties (35,36) and permeability properties (32, 37) of connective tissues. The DS-protein isolated in this study has markedly different physical properties and molecular weight to chondroit,in sulfate- and hyaluronate-proteins and it is probable that DS-prot’ein molecules do not interact to form an extensive net,work like the other two acid mucopolysaccharides (29). HoJvever, the difference in behavior between the DS-protein and chondroitin sulfate- or hyaluronateproteins may be due to their different modes of preparation, e.g., exposure to 6 M urea at 60”. DS-protein, but not the chondroitin sulfate- or hyaluronate-proteins of bovine heart valves, has been found to precipitate a fraction of tropocollagen instantaneously as “native-type” fibrils at physiological pH and ionic strength (Fig. 5). When the mixtures of DS-protein and tropocollagen were incubated at 37” until fiber formation was complete the resulting gels mere fibrous in appearance (Fig. 7). In addition the DSprot’ein is firmly associated with collagen fibers in skin and heart valves (3-5) and can only be fully extracted by gelatinizing or degrading a large proportion of fibers. It therefore seems likely that the primary biological role of DS-protein is in t,he formation and orientation of collagen fibers. ACKNOWLEDGMENTS The authors are most grat.eful t.o Dr. B. N. Preston for performing the free boundary electrophoresis, to Mr. B. Veitch for the electron microscopy, to Mr. G. Nicol for the amino acid analyses. to Mrs. A. Goh and Mr. F. A. Meyer for gifts of hyaluronateand chondroitin sulfate-proteins, and to Mr. J. Hllmphrey for t.echnical assistance. We also wish t.o thank t.he Australian Meat Research Organisat,ion and the National Heart Foundation of Australia for financial support. REFERENCES 1. MUIR, H., in “International Reviews of Connective Tissue Research” (D. A. Hall, ed.), Vol. 2, p. 101. Academic Press, New York (1964). 2. SCHUBERT, WI., Federation Proc. 26,1047 (1966).

577

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