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Acid hydrolysis of acidic mucopolysaccharides
MUCOPOLYSACCHARIDES
from
HYDROL!I’SIS
151
Chondroitin 4-sulfate was isolated from bovine cartilage powder, obtained Wilson Laboratories, Chicago, Illinois. The powder was stirred with IO parts
of 0.05 N NaOH (w/v) at 4” for 2 days and the mixture centrifuged. The supernatant fraction was neutralized with acetic acid and thrichloroacetic acid was added until a concentration of 4% was reached. After being kept for 30 min at room temperature, the mixture was centrifuged. In order to purify the polysaccharide, I g of Darco G-60 was added per 3 g of starting material. The relatively small amounts of polysaccharide adsorbed by charcoal were eluted by treating the charcoal at 4” for 4 or 5 h with 30% ethanol containing 0.5% NH40H. The charcoal supernatants and eluates were dialyzed and purified further by means of chromatography on Dowex-I. Anal.: Uranic acid, 29.0%; hexosamine, 24.5%; N, 2. I %. Dermatan sulfate was isolated from heparin by-products (obtained through the courtesy of the late Dr. A. Winterstein, Hoffmann-La Roche, Basel., Switzerland, and Dr. H. H. R. Weber, Wilson Laboratories, Chicago, Illinois) by use of a copper using two precipitations for the final product. A low color precipitation methodls, yield is normally obtained in the cronic acid estimation of dermatan sulfate when using the carbazole methodlg. Anal.: Uranic acid, 14.6%; hexosamine, 29.8%; N, 2.4%. Chondroitin 6-sulfate obtained from shark cartilage was a product of the Kaken-Yaks Kako Co., Ltd., Tokyo, Japan, and used without further purification. Anal.: Uranic acid, 31.0%; :lexosamine, 28.3%; N, 2.5%. Keratosulfate was isolated from human nucleus pulposus. The .,lcetone-dried tissue was digested with papain and the extract, kindly provided by Dr. A. Saunders. purified by use of charcoal and chromatography on Dowex-I. Anal.: Uranic acid, 1.4%; c;lrbohydrate, 22.7%; hexosamine, 22.8’:<; N. 2.h’:;. Heparin and heparitin sulf,ate were generously supplied by Dr. L. L. Coleman of the Upjohn Co., Kalamazoo, ‘Michigan, and Dr. H. H. R. Weber, Wilson Laboratories, Chicago, Illinois. and were: purified by the use of cetylpyridinium chloride and fractionation on Dowex-r as desl:ribed previouslys. High color yields fclr uranic acid estimations by the carbazole m&hod are normally obtained for both heparin and heparitin sulfate. Anal.: Uranic acid, 38.3%: hexosamine, 22. I %; total sulfate, 28.1%; N. 1.8%. 23.9%; N, 2.05%; total sulfate, Heparitin sulfate: Uranic acid, ~9.6%; hexosamine, 14.0”,; N-sulfate, 7.4%. N-Acetylheparin was prepared from 600 mg of heparin which had been desulfated by heating in 0.04 N IICl (IOO ml) for IOO min. After adjusting the pH to 8.0 by addition of I N NaOH, acetic anhydride (1.0 ml) and satura!:cd NaHCOs (5 ml) were added and the soluiion stirred occasionally over a 3o-min period. The pH was again adjusted to 8.0-8.5 and the acetylation procedure r,epeated with half the quantities of acetic anh!.dride and NaHCOs. Finally, the soluiion was concentrated in a flash evaporator 3t 35” and the N-acetylated heparin (550 mg) was Cnrboh_rdmreRes., z ( I 966) I so-- I 6 I
@pitated
by the addition of 3 vol. of alcohol, The nitrous acid-indole reaction14 indicated complete IV-acetylation. Anal‘: Uranic acid, 29.8%; hexosamine, 22.8%; N, 1.6%; sulfate, 19.4%.
Pnrpzuarl~ of oligosucchurides Oligrrsaccharides from chondroitin 4-sulfate and chondroitin Icsalfate were &Mkd rnfter exhaustive digestion with testicular hyaluronidases0 and purifmttion
a~1%p&adex or Iiowex-lt columns. An oligosaccharide preparation Fromchondroitin 4+tdfate, which had been fractionated on a Sephadex G-25 column, was kindly pmtided by Dr. A. Saunders. An&.: Uranic acid, 35.2%; hexosamine, 28.0%; reducing sugar, 11.4%. The products of chondroitin &sulfate digestion were fractionated on a Dowex-r e&~&n, The material eluted with I .o and I .5 M LiCl was concentrated and precipitated with 4 vol. of ethanol. A&.: Uranic acid, 36.8 %; hexosamine, 30.0%; IV-acetylhexosamine, 16.5 %_ NmAcertylchondrosinewas prepared by hydrolysing of chondroitin 4-sulfate with 1 H HCII for 2 h, adsorption of the chondrosine on charcoal, washing the adsorbent SW& times with water to remove acid and free hexosamine, and eluting of the chandrosinc with hot 30% ethanol. Acetylation was performed directly on the Amhcrl eluate by the addition of acetic anhydride and NaHC& as described for
P&mtyihcparin. And.:
Uranic
acid, 36.3 %; hexosamine,
30.8 %; IV-acetylhexosamine,
3 1.0%.
?fy&olysis conditions The: conditions used for determining the hydrolysis of mucopolysaccharides OS illustrated in Figs. I and 2 were as follows: solutions (0. I %) of the substances in OXI?to 0.2 N NC1 were heated in screw cap tubes with Teflon liners. After heating
PQ t Hcrk~e of reducing groups during hydrolysis of mucopolysaccharides. Values are expressed rrr~Wstim of reducing sugar to total hexosamine content. Conditions of hydroiyris a5 dzscriki m $%#uM#HTAL. Ahbroviations: HS, heparitin sulfate; AcHep, IV-acetyibeparin; KS, keratan sulfate; C,?M, derninlan sulfate; HA, hyaluronic acid; CS-C, chondroitin f&sulfate.
for 1 b, the hydrolyzates were co&d under tap water and neutralized by ‘the addition of eqtivaknt zmaunts of NB&OJ, AnaXyses were carried out on the neutralized solutions.
rlll,.*..l.l.-l
I..,
.,,,,,,,,,,.,,
, .,,,,,,,,,,.,,,
. . . . ..
c!j..c
..-*~HA
Fig. 2. Liberation of hexosamine reducitg units determined by the Morgan-Ebon reaction after hydrolysis of mucopolysaccharides as de&bed in EXPERIMENTAL. Values are cxpressc~i as ratios or N-acetyihexosamine to total hexaramiac: content. The solid line refers to results abtainecl after acetylatian of the hydrotyzates as describ~:clby Dewy and McAllanla, and the broken lines represent values obtained by direct analysis af the hydralyzates, For abbreviatioas, see Fig. I,
Following hydrolysis of mucopoiysaccharides with 0.04 M MCI, ?ree amino groups were estimated (Fig. 3) as described by Foster et aP2. One ml of o.o4 N hydro-
Fig. 3, ReIease of free amino e~roups from bexosamine during hydrolysis of t’mlcopolysaccharides periods up to 2 h. E;stimation af the liberated amino groups was accomplished by application afthe X-Buoro-2,4-clinitrobenzene method q All rf%Wltswere related to heparin used as a standard. For abbreviations, 51%Fig. I ; CS-A, cbondroitin &sulfate. with 0.04 N WC3 for
Curbuiwdrute
h’s,
2 ( 1946) I ~J.-I(6f
chloric acid containing x mg of mucopolysaccharides in a IO ml volnmetric fiask WM heated for go min in a boiling water bath. To the cooled solution 0.4 ml at +aturatcd WaHCO3 solution, o. I ml of z-fluoro-z,4-dinitrobenzene and I .5 ml of ethanol. wcro added. The mixture was shaken vigorously for 4 h and then extracted twice with ether to remove excess x-fluoro-2,4dinitrobenzcne reagent. After being acidified with I .o N NCI, the sohttion was again extracted with ether and diluted to IO ml with MUX. The optica den&& at 355 rnp of the reaction products were compared with that from a st;andard heparin preparation. or1 firtrat1on Wydrolyzates of mucopolysaccharides were obtained by heating 8o-100 mg of mucopolysaccharidesin 2gml of a.04 N NC1 for 4h. After beingcoaled, the hydrafmw wer@neutralized with I u NaQH, concentrated to 2 ml and applied to a I. I x mo cm wlumn of Sephadex G-25 (3o-80 microns, beaded form). Elution was carried out with 1 N NaCl. Fractions of 3 ml were collected at 3c-min intervals and, after analysis for uranic acid content, were pooled as indicated in Fig. 5. Results of the analyses of the pooled material. are shown in Table I.
F&urn I illustrates the release of reducing groups, as determined by the alkahnef&cyanide method ae, after hydrolysis of mucopolysaccharides at KXP for I h with o.oz to 0.2 N HCI, The mucopolysaccharides, hyaluronic acid, chondroitin 6-sulfa&z, dcrmatsn sulfate, and keratan sulfate, in which the 2-amino-2-deoxyhexopyranwyl n?riidues are @a-linked appear to be cleaved to appreciable cxteats (approximately nvcragc disaccharide length with 0.2 N HCl), whereas the mucopolysaccharidtv having z-Minked residues, iV-acetylheparin ;.nd heparitin sulfate. are much more resistant to hydrolysis. Separate experiments with chondroitin 4-sulfate indicate that hydrolysis of this substance parallels that of chondroitin 6-sulfate and hydrolyis of hcparin is similar to that of heparitin sAlfate. Appraisal of the actual extent of hydrolysis, however, must take note of tht: kct that partial degradation of oligosaccharides may occur under the alkalirxt: conditions of the reducing sugar method. This degradation km-eases as the oligowcchnride sire decreases and is most pronounced with (143) linked reducing unir.9. When the alkaline-ferricyanide method is applied to PI-acetylchondror+ine, N-atxrgilIry&&Monic acid, and turanose,reducing values are obtained which indicate extensive clcnvage of the (I 43) linkages, whereas maltose and melibiose wl&h contain I A- and r&linkcd units, respectively, have been found to give reducing values only one-third or less above theoretical. Considered on this basis, the results shown in fig. I s bt? aakcn to indicate that the hydrolysis products obtained are larger than su~cst& !3~ !he reducing-sugar data. This is borne out by fractionation of the hydrolyzati by $61filtration on Sephadex G-25 as described later.
MUCOPOLY$ACCHARJDJZ!3 HYDROLYSIS
155
z-Amino-%deoxyglucoside bond cleavage An additional approach for evaluating the extent of the a-amino-2-deoxy-/I-Dhexopyranosyl bond cleavage in mucopolysaccharides was based on the formation of hexosamine reducing groups during hydrolysis. Figure 2 shows the N-acetylhexosamine values obtained after hydrolysis of mucopolysaccharides as described under “hydrolysis conditions”. The broken lines refer to the results of the MorganElson reaction carried out directly on the hydrolyzate and the solid lines represent the results found with the same method applied after acetylation as described by Levvy and McAllanls. The latter technique measures both hexosamine and N-acetylated hexosamine reducing units present after hydrolysis while the former measures only acetylated hexosamine. Disaccharides from the B-band a-L-linked mucopolysaccharides have been found to give color yields in the Morgan-Elson reaction equivalent to 8o_goo/, of those obtained with standard 2-acetamido-z-deoxyglucose. On this basis, the data in Fig. 2 indicate that up to one-third of the hexosamine reducing end groups are deacetylated after hydrolysis with 0.2 N acid for I h. Factors which affect the estimation of a-amino-z-deoxyglucoside bond cleavage by the Morgan-Elson method include the extent of both monosaccharide formation and desulfation during hydrolysis. Substitution by sulfate at C-4 of the &amino-zdeoxygalactose unit in chondroitin +sulfate and dermatan sulfate causes inhibition of color production in the calorimetric reaction 9.24. Consequently, the presence of sulfated oligosaccharides in hydrolyzates of these mucopolysaccharides results in decreased ratios of N-acetylhexosamine to reducing sugar. A similar decreased ratio results from the formation of free 2-acetamido+deoxygalactose, since this amino sugar yields only a third the color obtained from an equivalent amount of 2-acetamido2-deoxyglucose used as a standard in the Morgan-Elson method. A comparison of the relatively low IV-acetylhexosamine values with the reducing sugar values for dermatan sulfate, as noted in Figs. J and 2, suggests that this factor is of significance in the estimation of hexosaminidic bond cleavage for this mucopolysaccharide. Keratan sulfate, which is reported to contain O-galactosyl-( I--+-4)z-acetamido2-deoxyglucose linka~es25, produces oligosaccharides which are not expected to react in the N-acetylhexosamine method. The findings in Fig. 2, therefore, indicate the extent of monosaccharide formation from keratan sulfate. Approximately one-third of the keratan sulfate is cleaved to monosaccharides with 0.2 N HCI. The low values shown in Fig. 2 for the mucopolysaccharides with X-D-linked residues conform with their relative resistance to acid hydrolysis and to the formation of products which contain princinally uranic acid at the reducing ends. Deacetylatim of mucopoiysaccharides T?x above described methods were not applicable for estimating N-dcacetylation of internal hexosamine units. Liberation of such amino groups ws estimated by reaction of the hydrolyzates with J-fluoro-2.4-dinitrobenzene”J, using heparin :IS :I standard or by use of the indole-nitrous acid methodl”. Figure 3 shows the results Cuvbuhydrote
Res., z (I 966)
I E;o- I6 I
I.04
VI
,I
0.97 1.10 I.17
111 IV V
20
I.04
.
Uranic
acid
11
I
Fraction
_._
_,,_
a17
0.83 0.51 0.33
0 0.26
__.
LkO?liC
-.I
I.09
0.29
1.20 1.06 I.11 1.08
..-..
20
ncid
0.32 0.30
St&ale
-
0.19
0.87 0.43 0.29
044
-
hexosumine
N-Acetyl-
_
__
_
_.
-
(_)b 0.49 0.52
-
Surfare
.
.
U?QRk
-
0.53
0.56 0.55
4.0 0.56 0.40
acid
__-_-_--7
0.14
0.56 0.25 0.24
0 0.07
N-Ace?+ hexosumine
0.12
Gb .0.17
Sulfate
-
c
157
MUCCWOLYSA CCHARIDES HYDROLY!;IS hydrolysis of mucopolysaccharidcs
with 0.04 N HCI for up to 2 h, conditions used
in the determination of N-sulfate groups of heparitin sulfate and heparin14p21. It is noteworthy that N-acetylheparin, though resistant to glycosidic cleavage, is deacetylated approximately 30% after hydrolysis for 2 h. Similar results were found for keratan sulfate, but hyahmmic a&$ dermatan sulfate, and chondroitin 4-sulfate were more resistant to deacetylaGon. The progressive release of amino groups from heparitin sulfati indicates that appreciable deacetylation occurs along with Ndesulfation. Hydrolysis of oligosaccharides In order to study the course of acid hydrolysis of oligosaccharides, certain of these were subjected to the actiol of 0.04 N HCl for up to 6 h. Figure 4 illustrates the results obtained with oligosaccharides from chondroitin 4-sulfate (CSA-0), chondroitin d&fate (CSC-O), ar:d N-acetylated chondrosine ((3-O).
n-Y OROLYSIS Fig.
U-OURS
of oligosaccharides *lth 0.04 N HCI for up to 6 h as described in EXPERIMENTAL solid lines refer to reducing sugar talues and the broken ljnes represent N-awtythexosamine results obtained atkr acetylation. Vah es am expressed in terms of ratios to hexosamine content.
4. Hydrolysis
The
For abbreviations
see Text.
Preparation GSA-O, obtainlxl after exhaustive digestion of chondroitin 4-sulfate testicular hyaluronidase, appeared by analysis to be of average hexasaccharide six. As noted earlier, the presez~e of ester suIfatP at C-4 of the acetylhexosamine reducing unit inhibits color form; tion in the Morgan-Elson reaction. This analytical method therefore provides a con lenient means for determiuing the extent of desulfation of CSA-0 during acid hyd *olysis. The resultsshown in Fig 4 indicate that hydrolysis of CSA-0 yields an approximately parallel increase of reducing sugar (solid line) and ZV-acetylhexosamine values (broken lines). This sugg,rts that desulfation occurs at approximately the same rate as giycosidic bond cleavage. After hydrolysis for 6 h the ratio of reducing sugar to total hexosarm‘ne is appro &naXely I .o suggesting the formation of disaccharide units to a large extent. This is supported by the increase in JV-acet)-lhexosamine with
Carboh~d~are Res., z r.1966)
150-16~
158
J. A. CWONELLl
content after hydrolysis for 6 h to two-thirds that of CS-0, considered to be IargeIy disaccharide in nature. Since IO% or less difference was found between the N-acetyikxosamine values obtained before or after acetylation of hydrolyzates, it is concluded that the primary product of hydrolysis was a desulfated and N-ace&la&d disaccharide. The hydrolysis of an oligosaccharide fraction from chondroitin 6-sulfate &XXI) is also shown in Fig. 4. The results are found to be similar to those for CSA-0, 310 ng that disaccharide formation from this oligosaccharide occurs also under mii acid conditions. The extent of desulfation cannot be estimated in the same way us CBA-0 since the Morgan-Elson reaction is not affected by the C-6 sulfate in this MB!Ml~.
Hydrolysis of N-acetylchondrosine (CS-0), the desulfated repeating unit of both chondroitin 4- and 6-sulfate, indicates that cleavage beyond the stage of dbaccharide does not readily occur. This is indicated by the results in Fig. 4 which show relatively small increases in reducing sugar values and essentially unchanged Pkcctylhcxosamine values during hydrolysis.
aprdrsitin 4- and 6-sulfate and dermatan sulfate were hydrolyzed for 4 h and the hydrolyzates subjected to gel filtrationon Sephadex
LIPLUENT
VOLUME-
with 0.04 N HCI G-25 coiumns2~
ml
t ~a ri Prectionation of hydrolyzates from chondroitin 4-sulfate and dermatan *se %phnctc~ O-25. Curves shown are based on uranic acid contents.
sulfat
3) gel fiba~n
MUCOF’OLYSACCHARlDES
The fractionation
HYDROLYE IS
patterns
obtairied
r59 from hydrolyzates
of chondroitin
d-sulfate and
dermatan sulfate are shown in Efg. 5. Chondroitin S-sulfate yielded a pattern, not shown in Fig. 5, which was simikir to that from chondroitin +&fate. Analyses of the gel filtration fractions are shown in Table I. Data are given as molar ratios to hexosarnfne. As indicated above, N-acetylhexosamine estimations of chondroitin 6-sulfate fractiom. are a direct measure of molecular size, but results for the chondroitin 4-sulfate and dermatan sulfate products cannot be used in this way except when reducing end i exosamine units are totally devoid of sulfate. Results of the Morgan-Ehon method for chondroitin d-sulfate indicate that the main fractions in decreasing proportions are di-, tetra- and hexa-saccharides, respectively (fractions III, IV ant V of Fig. 5). Reduction with borohydride of fractions IV and III gave losses in ?exosamine contents of 44 and 91x, respectively, with no loss in uranic acid, in agreement with the Morgan-Elson data. The slowest fraction (peak I) contains almost entireiy uranic acid. When this was examined by paper chromatography, only su’3stances moving similar to glucuronolactone and glucuronic acid were noted. Fraction II showed, after paper chromatography, the presence of a-amino-2-de>xygaractose, 2-acetamido-a-deoxygalactose, and glucuronolactone. The fractions obtained from chondroitin a-sulfate appear similar to those from chondroitin &&fate except for the lower N-acetylhexosamine values which probably are attributable to the sulfate remaining after hydrolysis. Hydrolysis of dermatan sulfate produces a relatively large Proportion of free uranic acid (fraction I), amounting to possibly 107; of the total uronlc acid, when allowance is made for the lower color yield*9 of the free uranic acid corn 3ared to that bound in the oligosaccharide fractions. Paper chromatography showed the presence only of idurono!actone and iduronic acid in this fraction.
Partial hydrolysis of mucopt*lysaccharides for the production of oligosaccharides has been generally carried out under relatively vigorous acidic conditions. Thus, the preparation of disaccharides from hyaluronic acid and chondroitin d-sulfate has been accomphshed by heating at 190’ in I N HCI for several ha*5. Under these conditions, complete deaeetylation and Cesullation occurs. The present study indicates that mild acid conditions result in the extensive hydrolysis of p-o and a-L-linked mucopolysaccharides to produce mixtures c f oligosaccharides and monosaccharides which to a major extent remain acetyilated and which, except for hyaluronic acid products, are partially sulfated as well. The possibility of isolating such fragments is of obvious interest for structural studies. The exults from reducing sugar and N-acetylhexosamine predeterminations illustrated in Figs I and 2 suggest that small oligosaccharides ponderate after hydrolysis of the $-o-linked mucopolysaccharides with 0.1 or 0.2 s HCl for I h. the r-c+tind ed mucopolysaccharides, heparitin sulfate and As expected. Curbo~xdrufe
Rer..
2
(1966) I so-161
160
I.
A. CTFGYELU
IV-acetyiheparin, showed onlyaslight tendency for gfycosidic bond clcavageandermfld conditions. Despite this resistance to hydrolysis, N-acetylheparin was 30% deaaztylated (Fig. 3) when heated in o-o4 N HCI for 2 h, conditions used for the determination of N-sulfate groups in heparin and heparitin sulfatesl. The finding that heparitin sulfate, which contains both N-sulfateandWacety1 groqqshows a significam increase in reiease of amino groups beyond the time required for complete N-desulfarion indicates that the IV-acetyl bonds present in heparitin sulfate are as Iabile to acid as those found in N-acetylheparin and is another indication of the similarity of &z EWO substances. It is evident from the above results that determinations of N-sulfate contents by the methods generally used should be interpreted with caution. Sulfated oligosaccharides from chondroitin 4- and 6-sulfate, produced after exhaustive digestion of the mucopolysaccharides with testicular hyahuonidase were found to foIlow a course of hydrolysis similar to the parent mucopolysaccbaride. shown in Fig. 4. The hydrolysis yielded preponderantly disaccharide wbicb were relntively resistant to further cleavage under the conditions used. The isolation of oligosaccharide fractions from mucopolysaccharide bydrolyzntes obtained by heating with 0.04 N HCI for 4 h served to support the suggestion from Figs. I and 2 of the occurrence of extensive hydrolysis. As indicated in Table I. hydrolyzates from chondroitin 4- and &sulfate and dermatan sulfate contain disaccharides as major components, with larger oligosaccharides comprising successively smaller proportions of the product (Fig. 5). Free uranic acids were demonstm&d from each mucopolysaccharide. It is noted that the fractions have not been deacetylnted significantly. Desulfation was found to be more extensive in cbon&ottin d-sulfate and dermatan sulfate, which retained only a third or less of that ori@raill; present. than in chondroitin f$sulfate, which lost approximately haif its sulfate ACKNOWLEDGMENTS
We thank Dr. A. Dorfman and Dr. L. Roden for aid in the preparat;;~r of t mnnuscript. This work was supported by research grants from the Nationa instinnc of Arthritis and Metabolic Diseases, National Institutes of HeaIth, United Szates Public Hculth Service (grant AM-o5gg6-o4), and the Chicago Heart Association. JUMMARY
The hydroiysis of mucopolysaccharides with dilute hydrochsotic acid VI& stutlicd. Glycosidic bond cleavage of the /I-D- and a-~-linked mucopc+acchand~ ctr
( I
966) I 50-16 I
MUCOPOLYSACCHARIDES HYDROLYSIS
I61
remained relatively resistant to hydrolysis under the conditions used althpugh N-deaeetylation occurred more readily with N-acetylhepa~rin than with the o~her polysaccharides examined. OIigos.accharides of chondroitin 4- and 6-sulfate were found to undergo hydrolysis readily to N-aeetylated and partially sulfated disacqharides. REFERENCES 1 M. SATAKE AND H. MASAMUNE, J. ExptL Med., 68 (1958) 44. 2 M. L. WOLrROM, R . MONTGOMERY, J. V. KARABINOS, AND P. RATHGEB, ,/',. Am. Chem. Soc., 72 (195o) 57963 J- A. CIFONELLI AND A . DORFMAN, J. Biol. Chem., 235 (196o) 3283. 4 E. A. DAVIDSON AND K . MEYER, 2". Am. Chem. Soc., 76 (1954) 5686. 5 B. WEISSMAN, M. M . RAPPORT, A. LINKER, AND K. MEYER, J. BioL Chem., '~:o5 (t953) zos. 6 J. E. JORPES AND S. GARDELL, Y. BioL Chem., 176,1948) 267 7 1. YAMASHINA, A c t a Chem. Scand., 8 (1954) 1316. 8 K . H . MEYER AND G . BALmN, Heir. Chim. Acta, 36 (I953) 597i 9 S. SuzuKI AND J. L. STROMINGER, J. Biol. Chem., z35 (196o) 2768. Io S. SuzuKI, J. BioL Chem., 235 (196o) 3580. I I A. LIr~KER, K. MEYER, AND P. HOFFMAN, J. Biol. Chem., 236 (1961) 983. 12 R. W. JEANLOZ AND D. A. JEANLOZ, Biochemistry, 3 (1964) 121. 13 G. A. LEvvY AND A . McALLAN, Biochem. J., 73 0 9 5 9 ) 127. 14 D. LAGUNOFF AND G . WARREN, Arch. Biochem. Bio]~hys., 33 (1962) 396. 15 M. DUnOlS, K. A. GILLES, J. K. HAMILTON, P. A. REBERS, AND F. SMITH, A hal. Chem., 28\(1956) 35o. 16 J. A. CIFONELL! AND M. MAYEDA, Biochim. Biophys. Acta, 24 (1957) 397. 17 S. SCHILLER, G. A. SLOVER, AND A. DORFMAN, J. BioL Chem., 236 (1961) 983. 18 J. A. CIFONELLI, J. LUDOW1EG, AND A. DORFMAN, J. BioL Cllem., 233 (1958) 54J19 P. HOFFMAN, A. LINKER, AND K. MEYER, Science, 124 (1956) 1252. 20 B. WE1SSMANN, K. MEYER, P. SAMPSON, AND A. LINKER, J. B;oL Chem., 208 (t954) 417-': 21 A. B. FOSTER, E. F. MARTLEW, AND M. STACEY, Chem. Ind. ( L o n d o n ) , (t953) 899. 22 J. T. PARK AND M. J. JOHNSON, J. Biol. Chem., 181 (1949) 149. 23 R. KUHN, J. J. BAER, AND A. G~UHE, Chem. Ber., 87 (x954) t553. 24 R. Kt:tt'~, A. GAt:ttE, AXD H. H. BatR, Chem. Ber., 87 (1954; 1138. 25 S. H1RANO, P. HOFFMAN, AND K. MEYER, J. Org. Chem., 26 (1961) 5064. 26 P. FLODIr~, J. D. GREGORY, AND L. ROOI~N, Anal. Biochem., 8 (1964) 424.
Carbohydrate Rex., 2 (1966) 15o 16t
from
HYDROL!I’SIS
151
Chondroitin 4-sulfate was isolated from bovine cartilage powder, obtained Wilson Laboratories, Chicago, Illinois. The powder was stirred with IO parts
of 0.05 N NaOH (w/v) at 4” for 2 days and the mixture centrifuged. The supernatant fraction was neutralized with acetic acid and thrichloroacetic acid was added until a concentration of 4% was reached. After being kept for 30 min at room temperature, the mixture was centrifuged. In order to purify the polysaccharide, I g of Darco G-60 was added per 3 g of starting material. The relatively small amounts of polysaccharide adsorbed by charcoal were eluted by treating the charcoal at 4” for 4 or 5 h with 30% ethanol containing 0.5% NH40H. The charcoal supernatants and eluates were dialyzed and purified further by means of chromatography on Dowex-I. Anal.: Uranic acid, 29.0%; hexosamine, 24.5%; N, 2. I %. Dermatan sulfate was isolated from heparin by-products (obtained through the courtesy of the late Dr. A. Winterstein, Hoffmann-La Roche, Basel., Switzerland, and Dr. H. H. R. Weber, Wilson Laboratories, Chicago, Illinois) by use of a copper using two precipitations for the final product. A low color precipitation methodls, yield is normally obtained in the cronic acid estimation of dermatan sulfate when using the carbazole methodlg. Anal.: Uranic acid, 14.6%; hexosamine, 29.8%; N, 2.4%. Chondroitin 6-sulfate obtained from shark cartilage was a product of the Kaken-Yaks Kako Co., Ltd., Tokyo, Japan, and used without further purification. Anal.: Uranic acid, 31.0%; :lexosamine, 28.3%; N, 2.5%. Keratosulfate was isolated from human nucleus pulposus. The .,lcetone-dried tissue was digested with papain and the extract, kindly provided by Dr. A. Saunders. purified by use of charcoal and chromatography on Dowex-I. Anal.: Uranic acid, 1.4%; c;lrbohydrate, 22.7%; hexosamine, 22.8’:<; N. 2.h’:;. Heparin and heparitin sulf,ate were generously supplied by Dr. L. L. Coleman of the Upjohn Co., Kalamazoo, ‘Michigan, and Dr. H. H. R. Weber, Wilson Laboratories, Chicago, Illinois. and were: purified by the use of cetylpyridinium chloride and fractionation on Dowex-r as desl:ribed previouslys. High color yields fclr uranic acid estimations by the carbazole m&hod are normally obtained for both heparin and heparitin sulfate. Anal.: Uranic acid, 38.3%: hexosamine, 22. I %; total sulfate, 28.1%; N. 1.8%. 23.9%; N, 2.05%; total sulfate, Heparitin sulfate: Uranic acid, ~9.6%; hexosamine, 14.0”,; N-sulfate, 7.4%. N-Acetylheparin was prepared from 600 mg of heparin which had been desulfated by heating in 0.04 N IICl (IOO ml) for IOO min. After adjusting the pH to 8.0 by addition of I N NaOH, acetic anhydride (1.0 ml) and satura!:cd NaHCOs (5 ml) were added and the soluiion stirred occasionally over a 3o-min period. The pH was again adjusted to 8.0-8.5 and the acetylation procedure r,epeated with half the quantities of acetic anh!.dride and NaHCOs. Finally, the soluiion was concentrated in a flash evaporator 3t 35” and the N-acetylated heparin (550 mg) was Cnrboh_rdmreRes., z ( I 966) I so-- I 6 I
@pitated
by the addition of 3 vol. of alcohol, The nitrous acid-indole reaction14 indicated complete IV-acetylation. Anal‘: Uranic acid, 29.8%; hexosamine, 22.8%; N, 1.6%; sulfate, 19.4%.
Pnrpzuarl~ of oligosucchurides Oligrrsaccharides from chondroitin 4-sulfate and chondroitin Icsalfate were &Mkd rnfter exhaustive digestion with testicular hyaluronidases0 and purifmttion
a~1%p&adex or Iiowex-lt columns. An oligosaccharide preparation Fromchondroitin 4+tdfate, which had been fractionated on a Sephadex G-25 column, was kindly pmtided by Dr. A. Saunders. An&.: Uranic acid, 35.2%; hexosamine, 28.0%; reducing sugar, 11.4%. The products of chondroitin &sulfate digestion were fractionated on a Dowex-r e&~&n, The material eluted with I .o and I .5 M LiCl was concentrated and precipitated with 4 vol. of ethanol. A&.: Uranic acid, 36.8 %; hexosamine, 30.0%; IV-acetylhexosamine, 16.5 %_ NmAcertylchondrosinewas prepared by hydrolysing of chondroitin 4-sulfate with 1 H HCII for 2 h, adsorption of the chondrosine on charcoal, washing the adsorbent SW& times with water to remove acid and free hexosamine, and eluting of the chandrosinc with hot 30% ethanol. Acetylation was performed directly on the Amhcrl eluate by the addition of acetic anhydride and NaHC& as described for
P&mtyihcparin. And.:
Uranic
acid, 36.3 %; hexosamine,
30.8 %; IV-acetylhexosamine,
3 1.0%.
?fy&olysis conditions The: conditions used for determining the hydrolysis of mucopolysaccharides OS illustrated in Figs. I and 2 were as follows: solutions (0. I %) of the substances in OXI?to 0.2 N NC1 were heated in screw cap tubes with Teflon liners. After heating
PQ t Hcrk~e of reducing groups during hydrolysis of mucopolysaccharides. Values are expressed rrr~Wstim of reducing sugar to total hexosamine content. Conditions of hydroiyris a5 dzscriki m $%#uM#HTAL. Ahbroviations: HS, heparitin sulfate; AcHep, IV-acetyibeparin; KS, keratan sulfate; C,?M, derninlan sulfate; HA, hyaluronic acid; CS-C, chondroitin f&sulfate.
for 1 b, the hydrolyzates were co&d under tap water and neutralized by ‘the addition of eqtivaknt zmaunts of NB&OJ, AnaXyses were carried out on the neutralized solutions.
rlll,.*..l.l.-l
I..,
.,,,,,,,,,,.,,
, .,,,,,,,,,,.,,,
. . . . ..
c!j..c
..-*~HA
Fig. 2. Liberation of hexosamine reducitg units determined by the Morgan-Ebon reaction after hydrolysis of mucopolysaccharides as de&bed in EXPERIMENTAL. Values are cxpressc~i as ratios or N-acetyihexosamine to total hexaramiac: content. The solid line refers to results abtainecl after acetylatian of the hydrotyzates as describ~:clby Dewy and McAllanla, and the broken lines represent values obtained by direct analysis af the hydralyzates, For abbreviatioas, see Fig. I,
Following hydrolysis of mucopoiysaccharides with 0.04 M MCI, ?ree amino groups were estimated (Fig. 3) as described by Foster et aP2. One ml of o.o4 N hydro-
Fig. 3, ReIease of free amino e~roups from bexosamine during hydrolysis of t’mlcopolysaccharides periods up to 2 h. E;stimation af the liberated amino groups was accomplished by application afthe X-Buoro-2,4-clinitrobenzene method q All rf%Wltswere related to heparin used as a standard. For abbreviations, 51%Fig. I ; CS-A, cbondroitin &sulfate. with 0.04 N WC3 for
Curbuiwdrute
h’s,
2 ( 1946) I ~J.-I(6f
chloric acid containing x mg of mucopolysaccharides in a IO ml volnmetric fiask WM heated for go min in a boiling water bath. To the cooled solution 0.4 ml at +aturatcd WaHCO3 solution, o. I ml of z-fluoro-z,4-dinitrobenzene and I .5 ml of ethanol. wcro added. The mixture was shaken vigorously for 4 h and then extracted twice with ether to remove excess x-fluoro-2,4dinitrobenzcne reagent. After being acidified with I .o N NCI, the sohttion was again extracted with ether and diluted to IO ml with MUX. The optica den&& at 355 rnp of the reaction products were compared with that from a st;andard heparin preparation. or1 firtrat1on Wydrolyzates of mucopolysaccharides were obtained by heating 8o-100 mg of mucopolysaccharidesin 2gml of a.04 N NC1 for 4h. After beingcoaled, the hydrafmw wer@neutralized with I u NaQH, concentrated to 2 ml and applied to a I. I x mo cm wlumn of Sephadex G-25 (3o-80 microns, beaded form). Elution was carried out with 1 N NaCl. Fractions of 3 ml were collected at 3c-min intervals and, after analysis for uranic acid content, were pooled as indicated in Fig. 5. Results of the analyses of the pooled material. are shown in Table I.
F&urn I illustrates the release of reducing groups, as determined by the alkahnef&cyanide method ae, after hydrolysis of mucopolysaccharides at KXP for I h with o.oz to 0.2 N HCI, The mucopolysaccharides, hyaluronic acid, chondroitin 6-sulfa&z, dcrmatsn sulfate, and keratan sulfate, in which the 2-amino-2-deoxyhexopyranwyl n?riidues are @a-linked appear to be cleaved to appreciable cxteats (approximately nvcragc disaccharide length with 0.2 N HCl), whereas the mucopolysaccharidtv having z-Minked residues, iV-acetylheparin ;.nd heparitin sulfate. are much more resistant to hydrolysis. Separate experiments with chondroitin 4-sulfate indicate that hydrolysis of this substance parallels that of chondroitin 6-sulfate and hydrolyis of hcparin is similar to that of heparitin sAlfate. Appraisal of the actual extent of hydrolysis, however, must take note of tht: kct that partial degradation of oligosaccharides may occur under the alkalirxt: conditions of the reducing sugar method. This degradation km-eases as the oligowcchnride sire decreases and is most pronounced with (143) linked reducing unir.9. When the alkaline-ferricyanide method is applied to PI-acetylchondror+ine, N-atxrgilIry&&Monic acid, and turanose,reducing values are obtained which indicate extensive clcnvage of the (I 43) linkages, whereas maltose and melibiose wl&h contain I A- and r&linkcd units, respectively, have been found to give reducing values only one-third or less above theoretical. Considered on this basis, the results shown in fig. I s bt? aakcn to indicate that the hydrolysis products obtained are larger than su~cst& !3~ !he reducing-sugar data. This is borne out by fractionation of the hydrolyzati by $61filtration on Sephadex G-25 as described later.
MUCOPOLY$ACCHARJDJZ!3 HYDROLYSIS
155
z-Amino-%deoxyglucoside bond cleavage An additional approach for evaluating the extent of the a-amino-2-deoxy-/I-Dhexopyranosyl bond cleavage in mucopolysaccharides was based on the formation of hexosamine reducing groups during hydrolysis. Figure 2 shows the N-acetylhexosamine values obtained after hydrolysis of mucopolysaccharides as described under “hydrolysis conditions”. The broken lines refer to the results of the MorganElson reaction carried out directly on the hydrolyzate and the solid lines represent the results found with the same method applied after acetylation as described by Levvy and McAllanls. The latter technique measures both hexosamine and N-acetylated hexosamine reducing units present after hydrolysis while the former measures only acetylated hexosamine. Disaccharides from the B-band a-L-linked mucopolysaccharides have been found to give color yields in the Morgan-Elson reaction equivalent to 8o_goo/, of those obtained with standard 2-acetamido-z-deoxyglucose. On this basis, the data in Fig. 2 indicate that up to one-third of the hexosamine reducing end groups are deacetylated after hydrolysis with 0.2 N acid for I h. Factors which affect the estimation of a-amino-z-deoxyglucoside bond cleavage by the Morgan-Elson method include the extent of both monosaccharide formation and desulfation during hydrolysis. Substitution by sulfate at C-4 of the &amino-zdeoxygalactose unit in chondroitin +sulfate and dermatan sulfate causes inhibition of color production in the calorimetric reaction 9.24. Consequently, the presence of sulfated oligosaccharides in hydrolyzates of these mucopolysaccharides results in decreased ratios of N-acetylhexosamine to reducing sugar. A similar decreased ratio results from the formation of free 2-acetamido+deoxygalactose, since this amino sugar yields only a third the color obtained from an equivalent amount of 2-acetamido2-deoxyglucose used as a standard in the Morgan-Elson method. A comparison of the relatively low IV-acetylhexosamine values with the reducing sugar values for dermatan sulfate, as noted in Figs. J and 2, suggests that this factor is of significance in the estimation of hexosaminidic bond cleavage for this mucopolysaccharide. Keratan sulfate, which is reported to contain O-galactosyl-( I--+-4)z-acetamido2-deoxyglucose linka~es25, produces oligosaccharides which are not expected to react in the N-acetylhexosamine method. The findings in Fig. 2, therefore, indicate the extent of monosaccharide formation from keratan sulfate. Approximately one-third of the keratan sulfate is cleaved to monosaccharides with 0.2 N HCI. The low values shown in Fig. 2 for the mucopolysaccharides with X-D-linked residues conform with their relative resistance to acid hydrolysis and to the formation of products which contain princinally uranic acid at the reducing ends. Deacetylatim of mucopoiysaccharides T?x above described methods were not applicable for estimating N-dcacetylation of internal hexosamine units. Liberation of such amino groups ws estimated by reaction of the hydrolyzates with J-fluoro-2.4-dinitrobenzene”J, using heparin :IS :I standard or by use of the indole-nitrous acid methodl”. Figure 3 shows the results Cuvbuhydrote
Res., z (I 966)
I E;o- I6 I
I.04
VI
,I
0.97 1.10 I.17
111 IV V
20
I.04
.
Uranic
acid
11
I
Fraction
_._
_,,_
a17
0.83 0.51 0.33
0 0.26
__.
LkO?liC
-.I
I.09
0.29
1.20 1.06 I.11 1.08
..-..
20
ncid
0.32 0.30
St&ale
-
0.19
0.87 0.43 0.29
044
-
hexosumine
N-Acetyl-
_
__
_
_.
-
(_)b 0.49 0.52
-
Surfare
.
.
U?QRk
-
0.53
0.56 0.55
4.0 0.56 0.40
acid
__-_-_--7
0.14
0.56 0.25 0.24
0 0.07
N-Ace?+ hexosumine
0.12
Gb .0.17
Sulfate
-
c
157
MUCCWOLYSA CCHARIDES HYDROLY!;IS hydrolysis of mucopolysaccharidcs
with 0.04 N HCI for up to 2 h, conditions used
in the determination of N-sulfate groups of heparitin sulfate and heparin14p21. It is noteworthy that N-acetylheparin, though resistant to glycosidic cleavage, is deacetylated approximately 30% after hydrolysis for 2 h. Similar results were found for keratan sulfate, but hyahmmic a&$ dermatan sulfate, and chondroitin 4-sulfate were more resistant to deacetylaGon. The progressive release of amino groups from heparitin sulfati indicates that appreciable deacetylation occurs along with Ndesulfation. Hydrolysis of oligosaccharides In order to study the course of acid hydrolysis of oligosaccharides, certain of these were subjected to the actiol of 0.04 N HCl for up to 6 h. Figure 4 illustrates the results obtained with oligosaccharides from chondroitin 4-sulfate (CSA-0), chondroitin d&fate (CSC-O), ar:d N-acetylated chondrosine ((3-O).
n-Y OROLYSIS Fig.
U-OURS
of oligosaccharides *lth 0.04 N HCI for up to 6 h as described in EXPERIMENTAL solid lines refer to reducing sugar talues and the broken ljnes represent N-awtythexosamine results obtained atkr acetylation. Vah es am expressed in terms of ratios to hexosamine content.
4. Hydrolysis
The
For abbreviations
see Text.
Preparation GSA-O, obtainlxl after exhaustive digestion of chondroitin 4-sulfate testicular hyaluronidase, appeared by analysis to be of average hexasaccharide six. As noted earlier, the presez~e of ester suIfatP at C-4 of the acetylhexosamine reducing unit inhibits color form; tion in the Morgan-Elson reaction. This analytical method therefore provides a con lenient means for determiuing the extent of desulfation of CSA-0 during acid hyd *olysis. The resultsshown in Fig 4 indicate that hydrolysis of CSA-0 yields an approximately parallel increase of reducing sugar (solid line) and ZV-acetylhexosamine values (broken lines). This sugg,rts that desulfation occurs at approximately the same rate as giycosidic bond cleavage. After hydrolysis for 6 h the ratio of reducing sugar to total hexosarm‘ne is appro &naXely I .o suggesting the formation of disaccharide units to a large extent. This is supported by the increase in JV-acet)-lhexosamine with
Carboh~d~are Res., z r.1966)
150-16~
158
J. A. CWONELLl
content after hydrolysis for 6 h to two-thirds that of CS-0, considered to be IargeIy disaccharide in nature. Since IO% or less difference was found between the N-acetyikxosamine values obtained before or after acetylation of hydrolyzates, it is concluded that the primary product of hydrolysis was a desulfated and N-ace&la&d disaccharide. The hydrolysis of an oligosaccharide fraction from chondroitin 6-sulfate &XXI) is also shown in Fig. 4. The results are found to be similar to those for CSA-0, 310 ng that disaccharide formation from this oligosaccharide occurs also under mii acid conditions. The extent of desulfation cannot be estimated in the same way us CBA-0 since the Morgan-Elson reaction is not affected by the C-6 sulfate in this MB!Ml~.
Hydrolysis of N-acetylchondrosine (CS-0), the desulfated repeating unit of both chondroitin 4- and 6-sulfate, indicates that cleavage beyond the stage of dbaccharide does not readily occur. This is indicated by the results in Fig. 4 which show relatively small increases in reducing sugar values and essentially unchanged Pkcctylhcxosamine values during hydrolysis.
aprdrsitin 4- and 6-sulfate and dermatan sulfate were hydrolyzed for 4 h and the hydrolyzates subjected to gel filtrationon Sephadex
LIPLUENT
VOLUME-
with 0.04 N HCI G-25 coiumns2~
ml
t ~a ri Prectionation of hydrolyzates from chondroitin 4-sulfate and dermatan *se %phnctc~ O-25. Curves shown are based on uranic acid contents.
sulfat
3) gel fiba~n
MUCOF’OLYSACCHARlDES
The fractionation
HYDROLYE IS
patterns
obtairied
r59 from hydrolyzates
of chondroitin
d-sulfate and
dermatan sulfate are shown in Efg. 5. Chondroitin S-sulfate yielded a pattern, not shown in Fig. 5, which was simikir to that from chondroitin +&fate. Analyses of the gel filtration fractions are shown in Table I. Data are given as molar ratios to hexosarnfne. As indicated above, N-acetylhexosamine estimations of chondroitin 6-sulfate fractiom. are a direct measure of molecular size, but results for the chondroitin 4-sulfate and dermatan sulfate products cannot be used in this way except when reducing end i exosamine units are totally devoid of sulfate. Results of the Morgan-Ehon method for chondroitin d-sulfate indicate that the main fractions in decreasing proportions are di-, tetra- and hexa-saccharides, respectively (fractions III, IV ant V of Fig. 5). Reduction with borohydride of fractions IV and III gave losses in ?exosamine contents of 44 and 91x, respectively, with no loss in uranic acid, in agreement with the Morgan-Elson data. The slowest fraction (peak I) contains almost entireiy uranic acid. When this was examined by paper chromatography, only su’3stances moving similar to glucuronolactone and glucuronic acid were noted. Fraction II showed, after paper chromatography, the presence of a-amino-2-de>xygaractose, 2-acetamido-a-deoxygalactose, and glucuronolactone. The fractions obtained from chondroitin a-sulfate appear similar to those from chondroitin &&fate except for the lower N-acetylhexosamine values which probably are attributable to the sulfate remaining after hydrolysis. Hydrolysis of dermatan sulfate produces a relatively large Proportion of free uranic acid (fraction I), amounting to possibly 107; of the total uronlc acid, when allowance is made for the lower color yield*9 of the free uranic acid corn 3ared to that bound in the oligosaccharide fractions. Paper chromatography showed the presence only of idurono!actone and iduronic acid in this fraction.
Partial hydrolysis of mucopt*lysaccharides for the production of oligosaccharides has been generally carried out under relatively vigorous acidic conditions. Thus, the preparation of disaccharides from hyaluronic acid and chondroitin d-sulfate has been accomphshed by heating at 190’ in I N HCI for several ha*5. Under these conditions, complete deaeetylation and Cesullation occurs. The present study indicates that mild acid conditions result in the extensive hydrolysis of p-o and a-L-linked mucopolysaccharides to produce mixtures c f oligosaccharides and monosaccharides which to a major extent remain acetyilated and which, except for hyaluronic acid products, are partially sulfated as well. The possibility of isolating such fragments is of obvious interest for structural studies. The exults from reducing sugar and N-acetylhexosamine predeterminations illustrated in Figs I and 2 suggest that small oligosaccharides ponderate after hydrolysis of the $-o-linked mucopolysaccharides with 0.1 or 0.2 s HCl for I h. the r-c+tind ed mucopolysaccharides, heparitin sulfate and As expected. Curbo~xdrufe
Rer..
2
(1966) I so-161
160
I.
A. CTFGYELU
IV-acetyiheparin, showed onlyaslight tendency for gfycosidic bond clcavageandermfld conditions. Despite this resistance to hydrolysis, N-acetylheparin was 30% deaaztylated (Fig. 3) when heated in o-o4 N HCI for 2 h, conditions used for the determination of N-sulfate groups in heparin and heparitin sulfatesl. The finding that heparitin sulfate, which contains both N-sulfateandWacety1 groqqshows a significam increase in reiease of amino groups beyond the time required for complete N-desulfarion indicates that the IV-acetyl bonds present in heparitin sulfate are as Iabile to acid as those found in N-acetylheparin and is another indication of the similarity of &z EWO substances. It is evident from the above results that determinations of N-sulfate contents by the methods generally used should be interpreted with caution. Sulfated oligosaccharides from chondroitin 4- and 6-sulfate, produced after exhaustive digestion of the mucopolysaccharides with testicular hyahuonidase were found to foIlow a course of hydrolysis similar to the parent mucopolysaccbaride. shown in Fig. 4. The hydrolysis yielded preponderantly disaccharide wbicb were relntively resistant to further cleavage under the conditions used. The isolation of oligosaccharide fractions from mucopolysaccharide bydrolyzntes obtained by heating with 0.04 N HCI for 4 h served to support the suggestion from Figs. I and 2 of the occurrence of extensive hydrolysis. As indicated in Table I. hydrolyzates from chondroitin 4- and &sulfate and dermatan sulfate contain disaccharides as major components, with larger oligosaccharides comprising successively smaller proportions of the product (Fig. 5). Free uranic acids were demonstm&d from each mucopolysaccharide. It is noted that the fractions have not been deacetylnted significantly. Desulfation was found to be more extensive in cbon&ottin d-sulfate and dermatan sulfate, which retained only a third or less of that ori@raill; present. than in chondroitin f$sulfate, which lost approximately haif its sulfate ACKNOWLEDGMENTS
We thank Dr. A. Dorfman and Dr. L. Roden for aid in the preparat;;~r of t mnnuscript. This work was supported by research grants from the Nationa instinnc of Arthritis and Metabolic Diseases, National Institutes of HeaIth, United Szates Public Hculth Service (grant AM-o5gg6-o4), and the Chicago Heart Association. JUMMARY
The hydroiysis of mucopolysaccharides with dilute hydrochsotic acid VI& stutlicd. Glycosidic bond cleavage of the /I-D- and a-~-linked mucopc+acchand~ ctr
( I
966) I 50-16 I
MUCOPOLYSACCHARIDES HYDROLYSIS
I61
remained relatively resistant to hydrolysis under the conditions used althpugh N-deaeetylation occurred more readily with N-acetylhepa~rin than with the o~her polysaccharides examined. OIigos.accharides of chondroitin 4- and 6-sulfate were found to undergo hydrolysis readily to N-aeetylated and partially sulfated disacqharides. REFERENCES 1 M. SATAKE AND H. MASAMUNE, J. ExptL Med., 68 (1958) 44. 2 M. L. WOLrROM, R . MONTGOMERY, J. V. KARABINOS, AND P. RATHGEB, ,/',. Am. Chem. Soc., 72 (195o) 57963 J- A. CIFONELLI AND A . DORFMAN, J. Biol. Chem., 235 (196o) 3283. 4 E. A. DAVIDSON AND K . MEYER, 2". Am. Chem. Soc., 76 (1954) 5686. 5 B. WEISSMAN, M. M . RAPPORT, A. LINKER, AND K. MEYER, J. BioL Chem., '~:o5 (t953) zos. 6 J. E. JORPES AND S. GARDELL, Y. BioL Chem., 176,1948) 267 7 1. YAMASHINA, A c t a Chem. Scand., 8 (1954) 1316. 8 K . H . MEYER AND G . BALmN, Heir. Chim. Acta, 36 (I953) 597i 9 S. SuzuKI AND J. L. STROMINGER, J. Biol. Chem., z35 (196o) 2768. Io S. SuzuKI, J. BioL Chem., 235 (196o) 3580. I I A. LIr~KER, K. MEYER, AND P. HOFFMAN, J. Biol. Chem., 236 (1961) 983. 12 R. W. JEANLOZ AND D. A. JEANLOZ, Biochemistry, 3 (1964) 121. 13 G. A. LEvvY AND A . McALLAN, Biochem. J., 73 0 9 5 9 ) 127. 14 D. LAGUNOFF AND G . WARREN, Arch. Biochem. Bio]~hys., 33 (1962) 396. 15 M. DUnOlS, K. A. GILLES, J. K. HAMILTON, P. A. REBERS, AND F. SMITH, A hal. Chem., 28\(1956) 35o. 16 J. A. CIFONELL! AND M. MAYEDA, Biochim. Biophys. Acta, 24 (1957) 397. 17 S. SCHILLER, G. A. SLOVER, AND A. DORFMAN, J. BioL Chem., 236 (1961) 983. 18 J. A. CIFONELLI, J. LUDOW1EG, AND A. DORFMAN, J. BioL Cllem., 233 (1958) 54J19 P. HOFFMAN, A. LINKER, AND K. MEYER, Science, 124 (1956) 1252. 20 B. WE1SSMANN, K. MEYER, P. SAMPSON, AND A. LINKER, J. B;oL Chem., 208 (t954) 417-': 21 A. B. FOSTER, E. F. MARTLEW, AND M. STACEY, Chem. Ind. ( L o n d o n ) , (t953) 899. 22 J. T. PARK AND M. J. JOHNSON, J. Biol. Chem., 181 (1949) 149. 23 R. KUHN, J. J. BAER, AND A. G~UHE, Chem. Ber., 87 (x954) t553. 24 R. Kt:tt'~, A. GAt:ttE, AXD H. H. BatR, Chem. Ber., 87 (1954; 1138. 25 S. H1RANO, P. HOFFMAN, AND K. MEYER, J. Org. Chem., 26 (1961) 5064. 26 P. FLODIr~, J. D. GREGORY, AND L. ROOI~N, Anal. Biochem., 8 (1964) 424.
Carbohydrate Rex., 2 (1966) 15o 16t