Isolation of Novel Disaccharides from Chondroitin Sulfates · Isolation of Novel Disaccharides from Chondroitin Sulfates * SAKARU Suzumt From the Department of Pharmacology, Washington

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 235, No. 12, December 1960

Printed in U.S. A.

Isolation of Novel Disaccharides from Chondroitin Sulfates *

SAKARU Suzumt

From the Department of Pharmacology, Washington University School of Medicine, St. Louis 10, Missouri

(Received for publication, March 28, 1960)

In a previous paper, the enzymatic transfer of sulfate from W-phosphoadenosine phosphosulfate to acetylgalactosamine monosulfate with formation of a radioactive product chromato- graphically identical to chemically synthesized acetylgalactosamine disulfate has been reported (1). Oligosaccharides with properties suggesting that they might contain polysulfated acetylgalactosamine were also isolated from hyaluronidase digests of enzymatically sulfated chondroitin sulfate A (2). These observations suggested the possibility that oligosaccharides bearing more than one sulfate residue per acetylgalactosamine residue might be isolated from naturally occurring chondroitin sulfate preparations.

Three types of chondroitin sulfate, termed ChS-At ChS-B, and ChS-C, are now recognized (3). The major repeating units of these compounds are believed to be (a) B-1,3-glucuronido- N-acetylgalactosamine-4-sulfate, (b) B-1,3-iduronido-N-acetylgalactosamine-4-sulfate, and (c) b-1,3-glucuronido-N-acetylgalactosamine-6-sulfate, respectively.2 In addition a chondroitin sulfate (which, for reasons which will become evident, will be termed ChS-D) has been isolated from shark cartilage (4, 5). This compound has an infrared spectrum identical to that of ChS-C (6), and it has been suggested that it is, therefore, identical to ChS-C isolated by Meyer et al. (7, 8) from human chon- drosarcoma. However, the unusually high sulfur content of ChS-D (7.6% which calculates to 1.3 residues of sulfate per acetylgalactosamine residueY and the nonidentity of the two polysaccharides as acceptors in enzymatic sulfation (9) suggested that they might be distinct polysaccharides.

Sulfated and nonsulfated disaccharides have been prepared from ChS-A, ChS-B, and ChS-D by digestion of the polysaccharides by an enzyme preparation from Proteus vulgaris, which contains both a chondroitinase and a chondrosulfatase (10). The characterization of these products will be reported in this paper.

* Supported by Grant A-1153 from the United States Public Health Service to Dr. Jack L. Strominger.

t Present address, Department of General Education, Nagoya University, Nagoya, Japan.

1 The abbreviations used are: ChS-A, ChS-B, and ChS-C, chondroitin sulfates A, B, and C; GalNAc, N-acetylgalactosamine: GalNAc-4S and GalNAc-6s. N-acetvlaalactosamine-4- sulfate and -6-sulfate. Abbreviations for disaccharides are indicated in the text.

* More exact chemical names for these compounds are (a) 3-O-(B-n-glucopyranosyluronic acid)-2-deoxy-2-acetylamino-n-galactose-4-sulfate, (b) 3-O-@-n-idopyranosyluronic acid)-2-deoxy- 2-acetyfamino-n-galactose-4-sulfate, and (c) 3-o-(@-D-glUCOpy-

ranosyluronic acid)-2-deoxy-2-acetylamino-n-galactose-6-sulfate, respectively. The shorter nomenclature (cf. 3) will, however, be used in the text.

3 Other chondroitin sulfate preparations contain between 0.6 and 0.9 sulfate residue per acetylgalactosamine residue (7).

EXPERIMENTAL PROCEDURE

Materials and Methods

Polysacchuride-ChS-A (11) and ChS-B4 (12) were kindly given by Dr. Roger Jeanloz. Chondroitin sulfate from shark cartilage (ChS-D) (4) was kindly given by Dr. F. Egami. The following analyses were obtained and calculated on a dry weight basis by allowing the sample of ChS-D to dry to a constant weight at 80” in a vacuum: (a) nitrogen by micro-Kjeldhahl, 2.51%; (b) sulfate-S, 7.57%; (c) galactosamine, 35.3%; (d) glucuronic acid, 37.8%; and (e) ash, 27.9%. The values were close to the theoretical values calculated for n~Cl,H,eO,rNSNaz except for the unusually high sulfate content (S/N, 1.32). By means of Dowex 50-H+ column chromatography, galactosamine was detected as a single hexosamine component and a crystal- line hydrochloride was obtained with m.p., 180”. Infrared spectrum of the chondroitin sulfate has been described by Nakanishi, Takahashi, and Egami (6).

Each of these preparations gave a single metachromatic band at the same position on paper electrophoresis (pH 7.0, 0.05 M phosphate).

Chondroitinase and Chondrosu2fatase-An acetone powder of P. vulgaris, Strain NCTC 4636, was kindly given by Dr. B. Spencer. A crude extract of this powder was prepared by the method described by Dodgson, Lloyd, and Spencer (14) (preparation B), and was employed under the conditions described below.

Preparation of Oligosaccharides-One gram of the chondroitin sulfate preparation (ChS-A, ChS-B, or ChS-D) was incubated in 100 ml of 0.08 M phosphate buffer, pH 7.8, containing 5 mmoles of NaF and the extract from 2.5 g of P. oulgaris acetone powder. Phosphate and fluoride inhibited the chondrosulfatase in the preparation without affecting the chondroitinase. The mixture was incubated at 37” for 10 or 24 hours, acidified with acetic acid, and heated in a boiling water bath for 5 minutes. The precipitate was removed by centrifugation and discarded. The supernatant solution was run onto a column containing 15 g of Darco G-60 mixed with 15 g of Celite 535. The column was washed with 200 ml of water and then eluted with 200 ml of 5% aqueous pyridine. The eluate was reduced in volume on a rotating evaporator and then taken to dryness over Pz05 in a vacuum. The yields were 0.75 g, 0.60 g, and 0.75 g from ChS-A, ChS-B, and ChS-D, respectively. To separate the oligosaccharides, these powders were dissolved in 4 volumes of water

4 The preparation ChS-B was resistant to enzymatic hydrolysis by testicular hyaluronidase. Although a paper chromatogram (13) of a hydrolysate of this sample showed that the major uranic component was iduronic acid, some glucuronic acid was also detected in the hydrolysate.

3580

December 1960 S. Suzuki

GalNAc. 4s

ADi-4S ! ! Ga’N& ADi-4S ! ADi-6S

? ADi-diSs ADi-di SD

ORIGIN

ChS-A ChS-B ChS-0

FIG. 1. Paper chromatographic separation in solvent B of oligosaccharides obtained by digestion of ChS-A, ChS-B, and ChY-D with Proteus chondroitinase.

and chromatographed as bands (5i cm) on 8 sheets of Whatman Xo. 31151 filter paper in solvent 1~ (see below) for 48 hours. Guide strips were cut and oligosaccharides were located by aniline hydrogen phthalate (15) (Fig. 1). These compounds were eluted from the remainder of the chromatograms with water. After drying the eluates in a vacuum over l’*Or,, further purifications were carried out by precipitating the compounds with acetone from aqueous methanol solutions.

Paper Chromatography and Ektrophweais-Descending paper chromatography was carried out on Whatman So. 3hI.M filter paper in the following solvents: .\, isobutyric acid-O.5 N am- monia (5:3); B, n-butanol-acetic acid-water (50: 12:25).

Paper electrophoresis was carried out on Whatman Ko. 1 pitper in 0.05 M acetate buffer, pH 5.2, at a potential gradient of 15 volts per cm for 2 hours (16). All compounds could be detected with the anilinr hydrogen phthalata spray (15). -4s a consequence of the fact that a,P-unsaturated acids absorb ultraviolet light (17)) compounds containing A4,5-glucuronic acid were detected also by ultraviolet absorption photography (Fig. 2). Several compounds containing an unusual sulfate residue, presumably on A4,5-glururonic acid (see below), were visualized as yellow spots by heating the chromatogram at 130” for 10 minutes. The mechanism of this color reaction is un- known.

Periodate Otidation-The microtechnique for the dettrmina- tions of periodate consumption and formaldehyde liberation was described in a previous paper (18). Oxidations were carried out at 28” in acetate buffer, pH 4.5. Under these conditions, acetylgalactosamine consumed 5 moles of periodate in 1 to 2 hours with no further uptake during 22 hours,

To isolate periodate-resistant fragments of disaccharides, 10 pmoles of the disaccharide were dissolved into 2 ml of 0.2 M acetate buffer, pH 4.5, containing 100 pmoles of periodate and the misture was kept in the dark at room temperature until the consumption reached a maximum (cf. Fig. 6). The mixture was then put on Whatman No. 3MM filter paper as a thin line, 40 cm long, and chromatographed in solvent B for 48 hours. Several guide strips were cut from the rhromatogram and sprayed with aniline hydrogen phthalate, and heated at 130” for 5 minutes. Fragments not destroyed by periodate were visualized as yellow, brown, or purple bands. Periodate and

FIG. 2. Ultraviolet absorption print of electrophoretic strip with unsaturated disaccharides.

iodate were also located as brown bands (&A (mobility relative to glucuronic acid) 0.58 and 0.32, respectively). The fragments thus located were cluted from the chromatogram with water.

Infrared Spectroscopy-Spectra were obtained with KBr pel- lets in a Perkin-Elmer model 21 spectrophotometer with a SaCl prism. The amount of sample was 0.1 pmole and 905; ethanol was used to dissolve it.

Other Analytical Procedrcres-Elementary analyses were performed by P. Mansour, Zurich, Switzerland. Methods for measurements of amino sugar and reducing end group have been described previously (1). A4,5-Glucuronic arid was measured by the Dische carbazole reaction (19), in which test its extinction coefficient is similar to that of glucuronic acid (17).

The procedure for determination of sulfate (20) was modified to a semimicro srale so that the final volume of colored solution was 2.5 ml. In the range of 3.5 to 11 ng of sulfate the standard curve prepared in this way is linear.

Bromine uptake was measured as follows: about 0.4 pmole of sample was dissolved in 500 ~1 of methanol. 100 ~1 of 0.1 nr bromine solution in chloroform were then added. After stand-

3582 Novel Disaccharides from Chondroitin Sulfates Vol. 33.3, so. 12

TABLE I Analytical data

Preparations

Molar rati;;;glucuronic / Ratio of re- Rp in

solvent A RCA in

Electro- ducmg ,voup solvent n phoretic . -’ to tote.1

I mobility

! Galactos- 1 galactos-

I amine ~~~___

C”,

16.0 0.98 28.0 0.88 16.0 0.98 28.0 0.92

j 28.0 0.94 28.0 1.00 39.0 1.02 39.0 0.90

Sulfate amine

___-

IX-OS IX-#

ADi-OS ADi-4S ADi-6S AIli-mono& ADi-diSB ADi-diSD

0.16 0.81 0.30 0.42 0.59 1.40

0.40 : 0.63 0.35 0.63 0.33 0.63

0.21 0.31 0.18 0.31

ersd mp in I Morgan-Elson 1 ha2 mp

reaction

/

0.00 1 1.25 13,000 200 0.00 0.39 0.96 0.90 l,ooO 200 0.00 0.13 0.00 1.09 15,ooo 7,900 1.10 0.36 0.92 ! 0.56 800 6,700 1 0.78 0.07 0.99 I 0.82 ’ / 15,000 7,200 0.99 0.03 1.10

j 0.98 / 13,000 6,900 0.7-I 0.55

2.08 0.47 1,300 0.57 1.90 0.62 ~ 13,000 0.15

* Periodate oxidations were performed at pH 4.5 for 20 hours. At pH 7.5, the values were about twice as high.

ing for I5 minutes in the dark, thr uptake of bromine was meas- dase digests of ChS-;\. The isomerir monosulfated unsaturated ured by iodometric titration. The titration value was sub- disacscharidcs were separated in solvent A as were the two di- tracted from a reagent blank. sulfated disaccharides (Fig. 3).

RESULTS

The following compounds have been isolated from the original ehondroitinase digests of the various polysaccharidcs (Fig. 1): (a) A4,5-glucuronido-acetylgalactosaminr (ADi-OS) from each of the polysaccharides (yield, about 400 mg per g of original polysacacharide) ; (b) A4,5-glucuronido-acetylgalactosamine-4- sulfate (ADi-4s) from ChS-A and ChS-I< (yield, 100 to 200 mg) ; (c) A4,5-glucuronido-acctylgalactosamine-6-sulfate (ADi- 6s) from ChS-D (yield, 100 to 200 mg); (d) a A4,5-glucuronido- acetylgalactosamine-6-sulfate bearing a second sulfate residue believed to be on the glucuronic acid moiety (ADi-di&) from ChS-D (yield, 25 mg). ;\ sulfate residue could be removed enzymatically from this compound to give a product, ADi- monoSn (an isomer of ADi-6S and ADi-4S), believed to contain a sulfate residue on the glucuronir acid moiety. Finally, (e) a disulfated A4,5-glucuronido-acetylgalactosamine (ADi -di&) from ChS-U (yield, 10 mg), distincst from ADi-diSD.5 The evidence for these assignments follows.

.Analytical Data-The ratios of galactosamine and sulfate to glucuronic acid were close to the theoretical values for the proposed structures (Table I).

The ratio of reducing groups to galactosamine indicated that ADi-OS could not be larger than the disaccharide. ADi-4S and ADi-6S have lower reducbing values than ADi-OS, presumably due to the sulfate substituent. As previously observed (18), sulfation depresses the reducing value of GalSAc, sulfation at the 4-position resulting in a greater depression than sulfation at the 6-position. The high reducing value of ADi-monoSD suggests that the acetylgalartosamine moiety in this compound is not substituted with sulfate.

Each of the compounds was hydrolyzed in 6 N HCl at 100’ for 15 hours. The resulting amino sugar was degraded with ninhydrin to a pentose which was identified by paper chromatography (21). Lyxose was the only pentose observed and hence each of the disaccharides contained only acetylgalactosamine.

Chromatographic and Electrophoretic Jlobilities-The monosulfated disaccharides migrated electrophoretically almost 2 times as fast as the nonsulfated disaccharides, and disulfated disaccaharides at almost 3 times the rate of the nonsulfated compounds (Fig. 2). These data are compatible with structures containing 1, 2, and 3 net negative charges. However, the nonsulfated disaccharides (ADi-OS and glucuronido-acetylgalactosamine (Di-OS)) had the same electrophoretic mobilities (Table I). Similarly, the monosulfated disaccharides (ADi-4S, ADi-GS, ADi-mono&,, and glucuronido-acctylgalactosamine-4- sulfate (Di-4s)) and the disulfated disaccharides (ADi-diS” and ADi-di&) had equal electrophoretic speeds.

The sample of ADi-OS was purified for analyses by repeating the precipitations from methanol with acetone and was dried _ _ in a vacuum over P205.

C,,Hs,OnN Calculated: C 44.33, H 5.58, N 3.69 Found : C 44.71, H 6.17, N 4.03

ADi-OS and ADi-4S have larger R, values in the two solvents employed than Di-OS and Di-4S (1) (which do not contain the A4,5-glucuronic acid moiety) previously isolated from hyaluroni-

Evidence for A-/t ,5- Cnsaturated Uranic Acid Component-Each of the oligosaccharides had the characteristic ultraviolet absorption spectrum of an a&unsaturated acid (Fig. 4). The es- tinction coefficient at 232 rnp, baaed on uranic acid content, was in each case about 7000 (Table I). This absorption of ultraviolet light permitted detection of the compounds on paper chromatograms under a “Mineralight” or by ultraviolet absorption photography as described by Markham and Smith (16) (Fig. 2).

Each of the compounds took up about 1 mole of bromine per mole of disaccharide (Table I). The acid lability of the uranic acid moieties in several cases (see below) also distin- guishes these compounds from the oligosaccharides containing a saturated glucuronic acid moiety.

d[urgan-Elson Reactions-The extinction coefficient at 585 rnp of GalNAc in the modified Morgan-Elson reaction (22) is

5 .4 small amount of ChS-C was kindly given by Dr. K. Meyer, and was also degraded to give three compounds with RF values corresponding to ADi-6S, ADi-OS, and GalNAc. Heparin was not hydrolyzed by this preparation. It should be especially noted that disulfated disaccharides were not observed with ChS-A, ChS-C or heparin.


9000. Di-OS, ADGOS, ADi-6S, ADi-diSn , and ADi-monoSo all have extinction coefficients in the range of 13 to 15,000, based on glucuronic acid (Table I). This value indicates (a) that the amino sugar is the reducing component of the disaccharide (since a free aldehyde is a requirement of the color reaction) and (5) that the size of the oligosaccharide cannot be larger than the disaccharide. The fact that the extinction coefficient is greater than that of Ga1N.k suggests that the a-position is substituted, presumably by the A4,5-glucuronic acid moiety. These high values also esclude substitution at thr 4-position which is known to inhibit greatly the color reaction (cf. 18). Di-4S, ADi-4S, and ADi-diSn have very low extinction coefficients (Table I), suggesting that the 4-position is substituted in earh of these corn- pounds.

.lniline Hydrogen Phthalate Color-ADi-OS, ADi-GS, ADi- diSn, and ADi-monoSn gave brown colors on paper chromatograms when sprayed with aniline hydrogen phthalate and heated at 130” for 5 minutes. ADi-4S and ADi-diSn gave purple colors. It has been shown previously that GalNAc and Gal- SAC-6S yield a brown color in this reaction whereas Ga1N.k4S yields a purple color (18). ADi-diSn , ADi-diSu , and ADi- mono&, also gave yellow colors without the reagent when heated at 130” for 10 minutes.

.tcid Hydrolysis-Treatment of ADi-4S and ADi-6S in 0.04 N HC1 at 100” for 60 minutes converted these compounds in a yield of over 89% to compounds chromatographkally and elec-

trophoretically identical to GalP\‘&+4S and GalNXc-GS, respectively. X complete characterization of these monosaccharides has already been presented (18). Under these conditions about 20yL of ADi-diSn was converted in 69 minutes to ADi-monoSo, with concomitant liberation of sulfate. ADi-monoSo was completely stable to acid hydrolysis, suggesting that this compound might contain a substituted A4,5-unsaturated glucuronic acid moiety.

Digestion by Chondrosulfatase-The crude enzyme prcpara- tion from P. wulgaris contains a chondrosulfatase (10). It was incubated with each of the sulfated unsaturated disaccharides in the absence of fluoride (a sulfatase inhibitor). ADG4S and ADi-6S were each converted to a compound with the electrophoretic mobility and analytical values of ADi-OS (Fig. 5). ADi-diSn was converted to a compound with the mobility of a

FIN. 3. Paper chromatogram developed in solvent A showing separation of all compounds. The spot due to IX-4S, which could

monosulfated disaccharide (ADi-monoSn). Sulfate analyses at not be seen in the photograph, is indicated by an arrow. x= several time intervals indicated that a maximum of 1.1 moles of artifact.

sulfate was released from ADi-diSn during the digestion. The . isolated product, ADi-monoSo, contained 1 sulfate residue (Table I) but was completely resistant to further hydrolysis by the Proteus extract (Fig. 5). Neither of the sulfate residues could be removed from ADi-diSB by this preparation (Fig. 5).

Periodate Oxidation-Although the absolute values for periodate consumption of such complex molecules are difficult to interpret, the differences between various compounds can be more readily interpreted. ADi-4S consumed 4 moles of periodate (Fig. 6). Since little or no formaldehyde was formed (Table I) during periodate oxidation and since GalNAc-4-S can 0.200 - be recovered after exhaustive periodate oxidation of ADi-4S (see below), the 4 moles of periodate consumed presumably I

represent the periodate consumption of the A4,5-glucuronic acid 220 240 260 7‘3

moiety. It has been noted previously that sulfation at the 4- WAVELENGTH, mp

_ position of GalNAc stabilizes the pyranose ring to some extent FIG. 4. Ultraviolet absorption spectrum of a disaccharide con-

so that formaldehyde liberation occurs more slowly than from taining A4,5-glucuronic acid (0.11 pmole of ADi-6S in 1 ml. of

GalNAk (18). Apparently, the substitution in ADi-4S at both water). Spectra of the other unsaturated disaccharides were essentially identical.

3584 Novel Disaccharides from Chondroitin Sulfates Vol. 233, so. 12

FIG. 5. Effect of chondrosulfatase on unsaturated disaccharides. To prepare the sample, 0.5 pmole of each disaccharide was incubated in 0.05 ml of 0.02 M phosphate, pH 7.0, with extract equivalent to 0.1 mg of acetone powder of Proteus vulgaris. After 8 hours, the incubation mixture was put on a small column containing 13 mg of Darco G-60. The column was then washed with 0.14 ml of water. The product was eluted from the column with 0.4 ml of 5% pyridine. This solution was dried in a vacuum, redis- solved in water, and subjected to paper electrophoresis. Under these conditions partial hydrolysis of AIli-4S, ADi-6S, and ADi- diSo occurred. With a longer incubation time complete hydrolysis was obtained. Compare with Fig. 2.

the 3- and 4-positions of GalS.\c stabilizes the ring almost completely. ADi-6S appears to be less stable to periodate since 1 or 2 moles of periodatc, in addition to those consumed by ADi- 4S, arc slowly taken up, with no formaldehyde formation. ADi-OS consumed 8 moles of periodatc with liberation of about 0.4 mole of formaldehyde.

ADi-diSo rapidly consumed 1 molt of periodate. However, the significance of this is obscure since the solution became cloudy during the titration and ADi-diSo could be recovered intact after 1 hour of oxidation by paper chromatography of the periodate incubation mixture. One more mole was con-

6 -AOi-OS

J ADi-6S

4 ,X---X t i IX --

T \ADi-4s

2

(52 AOi-diSg

d X!x

h x

= I / ADi-diSD 7

n 2 6 IO 14 22 40-56

TIME (HOURS)

FIG. 6. Rate of periodate consumption of unsaturated disaccharides. Kate the difference in the scale of the ordinate for the two sections of the figure.

sumcd slowly. ADi-monoSo, drrivrd from ADi-diSo by cn- zgmatic desulfation, consumed 2 additional moles of prriodate. .-\lthough no formaldehyde was formed from ADidiSo , 0.6 mole was formed from ADi-mono& indicating that the sulfate removed by sulfatasc had been linked to the primary alcohol group at C-6 of GalShc. ADi-diSa consumed 2 moles of periodate rapidly, accompanied by liberation of 0.6 molt of formaldehyde.

It is important to note that the minimal periodate consumption of the A4,5-glucuronic acid moiety is 4 moles. Therefore, both ADidiSo and ADi-diSB must contain a substituted A4,5- glucuronic acid moiety. The substituent is presumably sulfate.

Period&e Resistant Fragments--If either ADi-OS, ADi-4S or ADi-6S was treated for 2 hours with periodate, the glucuronic acid moiety (as measured by the carbazole reaction of the products isolated by paper chromatography) was quantitatively destroyed. Chromatography of the reaction mixture in solvent B indicated the appearance of new products with ROA values of 2.01,0.97, and 0.97, respectively. These products reacted with 2,4-dinitrophenylhydrazine to give a hydrazone which gave a red color in alkali,6 and are presumed to be the formate esters of GalNbc, GalNhc-4S, and GalNhc-6s. During recovery and manipulation, these fragments of ADi-4S and ADi-6S further decomposed to products with RGA values of 0.75 and 0.75, which did not form hydrazones. .inalyses of the final products indicated sulfate to amino sugar values of about 1. Data have been presented previously (18) which identify these products &s GalNAc-4S and GalNAc-6s. The yields of thr monosacc*ha- ride sulfates were 40 to 507c of unsaturated disacrharidrs.

Chromatography of a 18hour periodate digestion mixture of ADidiSo resulted in recovery of only the unchanged compound. However, after 56 hours of digestion, a product was formed with RaA = 1.577 which contained sulfate and gave a positive carba-

6 To 0.2 rmole of sample was added 0.02 ml of 0.4% P,+dinitrophenylhydrazine in 2 N HCl. After a few minutes, 1 ml of 0.5% alcoholic KOH was added. The formation of a red color indicated the presence of a hydrazone.

7 This product did not react with 2,4-dinitrophenylhydrazine. A smaller amount of another product (RaA = 1.80) which did form a hydrazone was also detected.


ZOO” CH20H A D-OS

tOOH

A Di-6s

GaINAc- 6s

Z~~0ti

Gal NAC - 4s

FIQ. 7. Summary of reactions of ADi-4S and ADi-6S

zole reaction, but did not contain amino sugar. The same product was formed after a 56-hour digestion of ADi-mono&,.

The only conclusion to be drawn from these studies is that the A4,5-glucuronic acid moieties of ADi-OS, ADi-4S, and ADi-6S are rapidly destroyed by periodate whereas those in ADi-diSn and ADi-mono& are relatively resistant to periodate. By contrast, the amino sugar in ADi-4S and ADi-6S is relatively resistant to periodate. However, the amino sugar in ADi- mono& is susceptible to destruction by prolonged periodate oxidation.

These data then also suggest that one of the sulfates of ADi- diSn (and the sulfate of ADi-mono&) is located on the A4,5- glucuronic acid moiety. The occurrence of a hexosamine-free, carbazole-reactive fragment after periodate oxidation strongly supports this proposal.

The reactions of ADi-4S and ADi-6S including desulfation with chondrosulfatase, oxidation with periodate, and degrada- tion with acid are summarized in Fig. 7. Possible structure of ADi-diSn is shown in Fig. 8.

Infrared Spectroscopy-Infrared spectra of disaccharides containing A4,5-glucuronic acid all showed bands at 785, 860, 910, and 980 cm-l due to the double bond (Fig. 9). The sulfate absorption at 1230 cm-l was seen in all the sulfated compounds. It has been established previously that substitution of sulfate at the 4-position in GalNAc showed characteristic bands at 700, 890, and 920 cm-l whereas substitution at the 6-position gave bands at 775, 820, and 992 cm-’ (18). Although in A4,5-unsaturated disaccharides some of the sulfate bands overlapped with those of the double bond, bands at 700, 860, and 920 cm-1 in ADi-4S and bands at 775, 820, and 992 cm-l in ADi-6S and ADi-diSn were evident. Disappearance of the band at 992 cm-1 in ADidi& after enzymatic desulfation (cf. spectrum of ADi-mono&) supports the conclusion that the hydrolyzed sulfate group was at the 6-position of GalNAc. No new bands were evident in ADidiSe, ADi-diSn or ADi-mono& which

HO+ (

COOH lH20S03H

A Di-diS,

FIG. 8. Proposed structure of ADi-diSn

could be assigned to the novel sulfate residue in these compounds.

DISCUSSION

Hyaluronidases of bacterial origin, which have been obtained from Pneumococcus, Staphylococcus, Streptococcus, and Clostrid- ium species, degrade hyaluronic acid to A4,5-glucuronido-acetyl- glucosamine (23). However, various chondroitin sulfates are not degraded by these preparations. On the other hand an adaptive enzyme from Flavobactium heparinum does degrade ChS-A and ChS-C to yield unsaturated oligosaccharides (23). It has been reported previously that the Proteus chondroitinase degrades ChS-A to yield a single product, glucuronido-acetylgalactosamine-sulfate (N-acetylchondrosine sulfate) (10, 24, 25), the repeating unit of ChS-A. However, in this study it is clear that the major sulfated disaccharide produced by the action of thii enzyme on ChS-A, ChS-B or ChS-D is a sulfated A4,5- glucuronido-acetylgalactosamine. No trace of N-acetylchondrosine sulfate or any other saturated oligosaccharide has been found among the reaction products. The mechanism of action

3586 Novel Disaccharides from Chondroitin Sulfates Vol. 235? No. 12

of Pro&w chondroitinase is, therefore, similar to that of other the 4position of acetylgalactosamine in GalNAc-S from ChS-A, known bacterial hyaluronidases and chondroitinases. to the 4position in GalNAc-S from ChS-B, and to the 6-position

Previously, acetylgalactosamine sulfate has been isolated from in GalNAc-S from ChS-D through comparison with samples ChS-A, ChS-B, and ChS-D. The sulfate has been assigned to which had been characterized as GalNAc-4S and GalNAc-6S

WAVENUMSER IN CM-’ WAVENUMBER IN CM-’

WAVELENGTH IN M’ZCNS WAVELENGTH IN M’ZCNS

WAVENUMBER IN CM-’ WAVENUMBER IN CM-’ 100 100 2500 2500 2occ 2occ l5cc I500 locc locc 900 900 800 700 800 700

90 90 P4.5-6l”c”ron,do-N.AcCt)llpoloclormul P4.5-6l”c”ron,do-N.AcCt)llpoloclormul

WAVENUMBER IN CM-’

0’ 8 I 3 4 5 6 7 8 9 IO II I2 I3 I4 I5

WAVELENGTH IN MCRONS


IO0

20

IO

0

WAVELENGTH lN MCRC+l5

FIG. 9A FIQ. 9. Infrared Spectra


WAVENUMBER IN CM-’ 1004ooo 3000 I. 2500 I,. 2000 I BOO 700 1500 I I I I I 1000 I 900 8

go- A Di-diS,

3 4 WAVELENGTH IN MICRCNS

WAVENUMBER IN CM-’ 100. .4900. 3000 I’ 2500 I”. 2000 I l5Kl I 1000 900 BOO 700 . I I I . a t 8

go-

BO- A Di- monoSD

IO-

0 3 4 5 6 I~AVELENGTH 8 9 IO II 12 13 I4 I5 IN t.mOr6


A Di-di SB

3587

Fro. QB

(18). These data confirmed and extended earlier studies of the positions of the sulfate substituents (cf. 18). Data on the A4,5glucuronido-acetylgalactosamine sulfates, reported in the present paper, are in agreement with these assignments.

The most unusual feature of the present investigation is the isolation of disulfated disaccharides from ChS-B and from ChS-D, in amounts of 1% and 2.5% of these polysaccharides, respectively.* These compounds were detected in the Proteus

* However, although the polysaccharide preparations used were of high purity, the possibility of 2 to 5% contamination by another polysaccharide of the preparations used cannot be excluded. Hence the possibility that the disulfated disaccharides were derived from impurities which contained a very high proportion of disulfated disaccharides also cannot be excluded. The possibility that the disulfated disaccharides were formed by sulfate transfer during enzymatic digestion is unlikely since no such products were detected during digestion of ChS-A and ChS-C, and since there are other obvious differences between ChS-C and ChS-D (sulfate content and suitability as acceptor for mucopoly- saccharide sulfotransferase).

chondroitinase digests as compounds with a high electrophoretic mobility. Various data, presented above, indicate that each of these compounds is a A4,5-glucuronido-acetylgalactosamine bearing two sulfate residues. It has been tentatively concluded that the compound from ChS-D is A4,5-&joglucuronido-acetylgalactosamine-6-sulfate, bearing a novel sulfate residue on the 2 or 3 position of the A4,5-glucuronic acid moiety. Although less information could be obtained about the disulfated disaccharide from ChS-B due to the smaller amount available, it is clearly a compound distinct from ADidiW.

*With regard to the structure of ADi-diSs, its infrared spectrum, low Morgan-Elson reaction, and purple color with aniline hydrogen phthalate suggest that one of the sulfate residues may be on the 4-position of acetylgalactossmine. However, the fact that ADi-diSs is distinguished from ADiJlS by ready liberation of formaldehyde and by resistance of both sulfate residues to the Protern sulfatase is not entirely consistent with this proposal. The low periodate consumption of ADi-diSa further suggests that the glucuronic acid moiety in this compound is also substituted, pas- sibly by the second sulfate residue.

3588 Novel Disaccharides from Chondroitin Sulfates Vol. 235, Ko. 12

Compounds with high electrophoretic mobility, presumably containing more than one sulfate residue per disaccharide, have also been obtained by enzymatic sulfation of ChS-A (2), and a mechanism for formation of acetylgalactosamine disulfate has been demonstrated (1). The complexity of the study of enzymatic sulfation of mucopolysaccharides is illustrated by the occurrence of sulfate linked (a) to the 4-position of GalNAc in ChS-A and ChS-B, (a) to the 6-position of GalNAc in ChS-C and ChS-D, (c) to the amino group of glucosamine in heparin and heparitin, and (d) as shown here, probably to the 2 or 3 position of glucuronic acid in ChS-D.‘O

SUMMARY

1. By means of enzymatic digestion with a chondroitinase preparation from Proteus vulgar&, A4,5-glucuronido-acetylgalactosamine and its sulfate esters have been derived from chondroitin sulfates A and B and from a chondroitin sulfate prepared from shark cartilage (chondroitin sulfate D). The sulfated disaccharide derived from both chondroitin sulfate A and chondroitin sulfate B has been characterized as A4,5-glucuronido- acetylgalactosamine-4-sulfate, whereas that from chondroitin sulfate D is the 6-sulfate isomer.

2. A novel disaccharide bearing two sulfates has also been isolated from the digest of chondroitin sulfate D. One of the sulfates is substituted at the 6-position of the acetylgalactosamine residue. Various data, particularly a low periodate consumption and the resistance of the uranic acid moiety to both periodate and acid, suggests that the second sulfate residue is substituted at the 2- or a-position of the uranic acid. A similar but distinct disulfated disaccharide has been isolated from a chondroitin sulfate B preparation.

~cknour2edgment-Grateful acknowledgment is made to Dr. J. I,. Strominger for his valuable discussions during these studies.

10 A heparitin sulfotransferase has recently been separated by DEAE-cellulose chromatography from sulfotransferases for several other mucopolysa&harides, and evidence for further separability of these enzymes has been obtained (S. Suzuki, R. H. Threnn, and J. L. Strominger, in preparation).

1.

2.

3.

4.

5. 6.

7.

8. 9.

10. 11.

12.

13.

14.

15. 16. 17.

18.

19. 20. 21.

22.

23.

24. MARTINEZ, R. J., WOLFE, J. B., AND NAKADA, H. I., J. Bac- teriol., 78, 217 (1959).

25. DODGSON, K. S., Biochim. et Biophys. Acta, 86, 532 (1959).

REFERENCES

SUZUKI, S., AND STROMINGER, J. L., J. Biol. Chem., 236, 267 ww .

SUZUKI, S., AND STROMINGER, J. L., J. Biol. Chem., 236, 274 (1960).

HOFFMAN, P., LINKER, A., AND MEYER, K., Federation Proc., 17, 1078 (1958).

SODA, T., EGAMI, F., AND HORIGOME, T., J. Chem. Sot. (Ja- pan), 61, 43 (1949).

UTIMARU, T., Bull. Chem. Sot. (Japan), 18, 235 (1943). NAKANISHI, K., TAKAHASHI, N., AND EGAMI, F., Bull. Chem.

Sot. Japan, 29, 434 (1955). MEYER, K., DAVIDSON, E., LINKER, A., AND HOFFMAN, P.,

Biochim. et Bio&vs. Acta. 81. 596 (1956). MATHEWS, M. B., iature (j&don), i81, 421 (1958). SUZUKI, S., AND STROMINGER, J. L., J. Biol. Chem., !Xl6, 257

(1960). DODGSON, K. S., AND LLOYD, A. G., Biochem. J., 88,88 (1958). EINBINDER, J., AND SCHUBERT, M., J. Biol. Chem., 191, 591

(1951). MARBET, R., AND WINTERSTEIN, A., Helv. Chim. Acta, 34,231l

(1951). CIFONELLI, J. A., LUDOWIEG, J., AND DORFMAN, A., J. Biol.

Chem., 288, 541 (1958). DODGSON, K. S., LLOYD, A. G., AND SPENCER, B., Biochem. J.,

66, 131 (1957). PARTRIDGE, S. M., Nature (London), 164, 443 (1949). MARKHAM, R., AND SMITH, J. D., Biochem. J., 62, 552 (1952). LINKER, A., MEYER, K., AND HOFFMAN, P., J. Biol. Chem., 219,

13 (1956). SUZUKI, S., AND STROMINGER, J. L., J. Biol. Chem., 286, 2678

wm DISCHE, Z., J. Biol. Chem., 167, 189 (1947). DODGSON, K. S., AND SPENCER, B., Biochem. J., 66,436 (1953). STOFFYN, P. J., AND JEANLOZ, R. W., Arch. Biochem. Biophys.,

62, 373 (1954). STROMINGER, J. L., PARK, J. T., AND THOMPSON, R. E., J.

Biol. Chem., 284, 3263 (1959). MEYER, K., LINKER, A., HOFFMAN, P., AND KORN, E. D.,

Proceedings of international symposium on enzyme chemistry, 1967, (Japan), Vol. 6, Maruzen Company, Ltd., Tokyo, 1958, p. 132.

Documents

Isolation of Novel Disaccharides from Chondroitin Sulfates · Isolation of Novel Disaccharides from Chondroitin Sulfates * SAKARU Suzumt From the Department of Pharmacology, Washington