On the association-dissociation of submaxillary mucin

81

NOTE.5

drofuran gel&t und in die Liisung 2-3 Stdn. aus einem Diboran-Generator Diboran (Trggergas: getrockneter Stickstoff) eingeleitet. Diboran wurde aus 5.1 g BFs-iitherat in 36 ml Diglyme durch Eintropfen von 25 mMo1 NaBHa in 20 ml Diglyme entwickelt. Anschliessend wurde ilberschilssiges Diboran mit Wasser-Tetrahydrofuran zersetzt und unter Kiihlung g ml 2~ NaOH und 4 ml Hz02 (30%) zugefilgt. Die Losung wurde i. Vak. zur Trockne eingeengt, der Riickstand in 25 ml Wasser aufgenommen und viermal mit 25 ml &her extrahiert. Nach Einengen des &hers verbleibt 1.8 g chromatographisch reiner Sirup, welcher beim Stehen kristallisierte. Umkrista!lisation aus Cyclohexan gibt 0.8 g (25%) II, Smp.6 g7.5O-g8”, [Q]E -35.3” (c 0.8, Methanol). Dilnnschichtchromatographie im Laufmittel Benz01 : Iithanol (3: 1) f 3% Wasser. Anal. Ber. filr C1eH2eOa:C 55.39 H 7.69. Gef.: C 55.19 H 7.80. H.

Chemisches Staatsinstitut, Institut fiir Organische Chemie, Unit~ersittit Hamburg (Deutschland)

PAULSEN

H. BEHRE

LITERATUR I z 3 4 5 6

H. C. BROWN, Nydroborurion, Benjamin, New York, rg6z. H. ZINNER, G. WULF, UND R. HEINATZ, Chem. Ber., g7 (1964) 3536. K. HEY~‘S IJND H. PAULSEN, Adcun. Carbohydrate Chem., 17 (1962) 169. M. L. Wousohl, J. Bmwshfmm, UND D. HORTON, J. Org. Chem. 27 (1962) 4505. J. LEHhlANN, Carbohydrate Res., 2 (1966) 1-13. D. C. DE JONGH UND K. BIEMANN, J. Am. Chem. Sot., 86 (1964) 67-

(Eingegangen

den 15. November,

1965) Curbohyakte

On the association-dissociation

of submaxillary

Res., 2 (1966) 80-81

mucin

Weight-average molecular weights between 4 x 106 and 8 x 106 have been reported for submaxillary mucins 122.The prevalent concept of the mucin structure is that the protein occupies the central core of the molecule to which are attached short, carbohydrate side-chains 394.Since the protein content of mucins is in the range from 37% [for bovine submaxillary mucins (BSM)] to 48% (for porcine submaxillary mucine), this would give a molecular weight of 1.5 x 10~ to 4 x IO* for the protein core. Cafbohyakxre Res., 2 (1966) 81-84

82

NOES

Proteins of very hi& molecuIar weight have been found to be aggregates of subunits, and most of the evidence supports the belief that single polypeptide chains having a molecular weight of greater than 6.6 x 10~ do not exis@. Urinary mucoproteins (mol. wt., 7 x 106) dissociate in urea to small subunits having molecular weights of 1.8 x 10s or less79s. Although BSM is polydisperse and contains1 a fraction having a molecular weight of 2 x 105, there remains the question of whether the fraction having a molecular weight of millions can be dissociated into smaller fragments by urea. To answer this, a BSM preparationla 2,5 was chromatographed on Sephadex G-200 (Pharmacia Lot No To-6471; particle size, 4o-120 p; water regain, 20 f 2 g) in aqueous 0.2~ sodium chloride and in 7~ urea containing 0.2~ sodium chloride. The length of the column was 55.5 cm and its volume was 201 ml. A 3.2-ml sample of an approximately I % solution of BSM was placed on the column equilibrated with 0.2~ sodium chloride. The mucin was eluted with 0.2M sodium chloride at a flow rate of approximately 4 ml/h, and 2-ml aliquots of the eluted samples were treated with 2 ml of Ehrlich’s reagent9; sialic acid was thereby used to indicate the presence of the mucin. A mixture of a O-I-ml sample of the original BSM solution, 1-9 ml of water, and 2 ml of Ehrlich’s reagent gave an optical density of 0.165 at 565 nyl. The chromatogram of BSM in 0.2~ aqueous sodium chloride (Fig. Ia) clearly shows the polydispersity of the BSM, as reported previouslyl. In order to obtain the void volume of the column, I ml of a I ok solution of dextran having a molecular weight of 2 x 106 (Pharmacia, FDR 922) was run on the same column under identical conditions. The eluted dextran was reacted with anthronelO, and the result is presented in Fig. ra. Similarly, 3 ml. of BSM and I ml of dextran (I % solutions) were chromatographed on Sephadex G-200 in 7~ urea and 0.2~ sodium chloride_ Theresults are given in Fig. Ib. The dextran gel swelled in urea and, therefore, less material was needed to fill the column. The flow rate could only be kept at approximately 0.5 ml/h_ The detection, with Ehrlich’s reagent, of the sialic acid-containing material in the eluate was done at 625 m,u(rather than at 565 m,uas in the aqueous solution) since the absorption maximum in urea solution occurs at this wavelength. Aliquots (2 ml) of the eluate were reacted with 2 ml of Ehrlich’s reagent. A mixture of a o. I-ml sample of the original BSM solution, 1.9 ml of urea solution, and 2 ml of Ehrlich’s reagent gave an optical density of 0.102 at 625 m,~c. In order to determine the swelling of the gel in urea, the column (packed in urea) was washed with 0.2M sodium chloride. and the void volume was redetermined with dexiran. It had increased from 78 to 91 ml, indicating a swelling of the gel grains in urea to 1.14 times their original volume in 0.2~ sodium chloride. The results of chromatography in the two media are relatively similar; the differences are a slight increase in elution volume for the mucin in urea compared to that in the aqueous medium and a more pronounced shoulder at an elution volume of 125 ml. The average distribution coefficient, Kav, between the gel phase and liquid phase of the BSM in aqueous medium was 0.19 and in 7~ urea was 0.23. These figures Carbohydrate

Res., 2 (1966) 81-84

83

NOTES

0.8

s

5 Ic g

0.6 0.4

if! 2

a2

e c G =

1.0

r” 2

0.8

d = y

0.6

0” 0.4 0.2

100

150

Fig. I. Chromatography of BSM (x) urea and 0.2hI NaCl (b).

ml and dextran (0) on Sephadex G-200 in

O.ZM

NaCl (a) and in

7hr

were calculated from the elution volumes (see ref. II) when half of the material had been eluted. This slight increase in the capacity of the gel in urea for BSM is of the order expected from the degree of swelling of the gel and is thus not a sign of a change in the molecular parameters of BSM. Ako, the more pronounced shoulder in Fig. rb when the chromatographic resolu . is explained by the change to a higher K arvalue tion increases. It was thus not possible to show a dissociation urea, as can be done for urinary mucoproteins73*.

of BSM

into smaller subunits

by

ACKNOWLEDGEMENTS

investigation was supported by a grant (HD-orqg) U.S. Public of Child Health and Human Development,

of the

This Institute

F.A.B.) and grants from the Swedish Medical Swedish Cancer Society (to T.C.L.).

Research

Council

Health

National

Service

(13x+ozA)

(to

and the

F. A. BEXTELHEIM* T. C. LAURENT

Deparmzent of Medical Chemistry, University of Uppsala (Sweden>

*Permanent address: Department of Chemistry, Adelphi University, Garden City, N.Y. Curbohydrufe

Res.,

2

(u. S. A.) (1966) 81-84

84

NOTES ,-

REFERENCES I 2

3 4 5 6 7 8 g IO II

F. A. B~LHEIM, Y.

HASHIhfOTO, AND W. PIGMAN, Biochim. Biophys. Acta, 63 (x962) 235_ F. A. BEI-X-ELHEIM AND S. K. DEY, Arch. Biochem. Biophys., 109 (1965) 259E. R. B. GRAHAM AND A. GOI-IXHALI~, Biochim. Biophys- Acta, 38 (1960) 513. A. GOTTSCHALKAND M. A. W. THOMAS, Biochim. Biophys. Acta, 46 (1961) 91. Y. HASHIMOTO,S. TSUIKI, K. NISIZAWA, AND W. Prch%m, Ann. N. Y. Acad. Sci., 106 (x963) 233. F. J. REITHEL, Adaan. Prorein Chem., 18 (1963) 123. C. C. CURTAIN, Australian J. Exptl. Biol. Med. Sci., 31 (1953) 615. M. M;UCFIELDAND M. S. DAVIES, Ann. N. Y. Acad. Sci., 106 (1963) 288. I. WERNER AND L. ODIN, Acta Sot. Med. Upsalien., 57 (1952) 230. R. DREYWOOD, Ind. Eng. Chem. (Anal.), 18 (1946) 499. T. C. LAURENT AND J. KILLANDER, J. Chromatog., 14 (1964) 317.

(Received December 6th, 1965) Carbohydrate Res.,

2

(1966) 81-84

Preliminary communications A new route to the synthesis of polysaccharides

Stereospecific synthesis of polysaccharides can serve as an important tool in chemical and biochemical investigations of these polymers. Syntheses already reported either do not give polymers of predicted structurel=2, or are not general method+s, or give rise only to oligomers 536. We now report a new route to the synthesis of polysaccharides having predictable types of glycosidic linkage. Sugar orthoesters, a new type of glycosylating reagent’, are used as starting materials. The new route is illustrated by the synthesis of an arabinan (I) containing predominantly a-( r+5)-L-arabinofuranosidic linkages, obtained by the polymerisation of #?-L-arabinofuranose r,2,5-orthobenzoate (IV).

The orthoester (IV) was synthesized as follows. Syrupy B-L-arabinofuranose 1,2(methyl orthobenzoate) 3,5-dibenzoate (II), [c& + rg” (chloroform), ng 1.5610, was saponified to give p-L-arabinofuranose r,2-(methyl orthobenzoate) (III), which reacted spontaneously to afford compound (IV), m-p. 14%r4g”, [a]~ + 30~ (chloroform) (Found: C, 61.3; H, 5.2; active H, 0.42. CiaHlaOs talc.: C, 61.0; H, 5.1; active H, 0.42%). Other tricyclic monosaccharide-orthoesters of type (IV) are known*Jg. Carbohydrate Res.,

2

(1966) 84-85