Dextran: Structure and Synthesis

DEXTRAN: STRUCTTTREAND SYNTHESIS BY W. BROCKNEELY Biochemical Research, Laboralor y , The Dow Chemical Company, Midland, Michigan I. Introduction.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Historical Review. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Source and Preparation of Dextran.. . . . . . . . . . . . . . . . . . . . . . . . IV. Structural Studies. . . . . . . . . . ............. 1. Chemical Studies.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................................. 2. Physical Studies.. . . . . . . . V. Enzymic Synthesis. . . . . . . . . .................................. 1. Acceptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Enzyme.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Mechanism of Polymerization ..................... .. VI. Uses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

341

344 349 356 357 361 363 364 366

I. INTRODUCTION The purpose of the present Chapter is to collect and disseminate the

information in the recent literature on dextran. The term dextran has been used for a class of D-glucose polysaccharides produced by bacteria growing on a sucrose substrate. I n this article, “dextran” will refer to those polysaccharides containing a backbone of D-glucose units linked predominantly + 6). The structural aspects of these polysaccharides, as revealed (Y-D-(~ by the earlier work, have been covered by Evans and Hibbert in a previous Volume of this Series.’ Their review will be brought up to date. In addition to describing the structural characteristics of dextran, a discussion of its enzymic synthesis has been included. It is the author’s hope that, on reaching the conclusion of this Chapter, the reader will have a deeper appreciation both of the enzymes capable of synthesizing dextran and of the polysaccharide itself. 11. HISTORICAL REVIEW Dextrans first came under close examination about a hundred years ago. The high viscosity of these slimes, as they were called, caused much trouble in the beet-sugar industries; the slimes blocked filters and interfered with the crystallization process. Scheibler2assigned the correct empirical formula (1) T. H. Evans and H. Hibbert, Advances in Carbohydrate Chem., 2,203 (1946). (2) C. Scheibler, Z . Ver. deut. Zucker-Ind., 19, 472 (1869); 24, 309 (1874). 341

342

W. BROCK NEELY

CeHloObfor this mucilaginous material; the molecular formula is now known to be ( C~ H ~ O O. ~Further, ), , he noticed that the material was closely related to starch and dextrin, and he therefore coined the name “dextran” for it. The early chemical and bacteriological work on dextran has been reviewed elsewhere.aThe work prior to 1937 suffered from study of impure preparations, and conflicting data resulted; this situation makes evaluation of that work diflicult. In 1937, however, Hibbert and coworkers‘ made the first systematic investigation of the chemical structure of dextran, and the work since that time has been on a much sounder basis. The ceI1-free enzymic synthesis of dextran from Leuconostoc mesenteroides was demonstrated by Hehre and Sugg.a,aThese two phases, the determination of structure and the enzymic synthesis of dextran, will be treated in greater detail in the ensuing pages of this article.

111. SOURCE AND PREPARATION OF DEXTRAN Hucker and Pederson: in 1930, classified the micro-organisms producing dextran (from sucrose) into Family, Tribe, Genus, and Species. This classification will be modified in the present Chapter, to conform to the nomenclature of Bergey? The main dextran-synthesizing bacteria belong to the Family Lactobmterimeae, Tribe Streptococceae, Genus Leuconostoc, Species L. mesenteroides (Betacoccus arabinosaceous) and L. dextranicum. The third species of Leuconostoc, namely, L. Citrovorum, does not produce dextran. In addition to these species, several other types have been shown to produce a polysacoharide similar to dextran. Bailey and Oxf0rd9J0have isolated a strain of Streptococcus bowis from the rumen which synthesizes dextran. Hehre” was able to demonstrate that a different strain of Streptococcus, DS strain 50, is capable of producing a dextran of low molecular weight. Betabacterium vermiformt5 was shown by Stacey and coworkers to synthesize (3) H. L.A. Tarr and H. Hibbert, Can. J . Research, B6, 414 (1931). (4) F. L. Fowler, I. K. Buckland, F. Brauns and H. Hibbert, Can. J . Research, B16, 486 (1937). (5) E. J. Hehre, Science, 93, 237 (1941). (6) E.J. Hehre and J. Y. Sugg, J . Exptl. Med., 75, 339 (1942). (7) G. J. Hucker and C. 8.Pederson, N . Y . State Agr. Ezpt. Sta. (Geneva, N. Y.), Tech. Bull. 167, 3 (1930). (8) R.8.Breed, E. G . D. Murray and A. P. Hitchens, “Bergey’s Manual of Determinative Bacteriology,” Williams & Wilkins Co., Baltimore, Md., 6th Edition, 1948. (9) R.W.Bailey and A. E. Oxford, J . Gen. Microbiol., 19, 130 (1968). (10)R.W.Bailey, Biochem. J . , 71, 23 (1959). (11) E.J. Hehre, J. Biol. Chem., %2, 739 (1956). (12) M. Stacey and F. R. Youd, Biochem. J . , 81, 1943 (1938). (13) W. D. Daker and M. Stacey, Biochem. J . , 82, 11 (1938).

DEXTRA N

343

a type of dextran,'* and, finally, ,Jeanes and her coworkers14demonstrated that two strains of Strqtobacterium dextranicum are capable of producing a polysaccharide similar to dextran. All of the aforementioned species have one characteristic in common, namely, that sucrose is the only suitable carbohydrate source for the manufacture, by them, of the polysaccharide. The organisms can grow on almost any medium containing sucrose together with a few inorganic salts and a suitable source of nitrogen. However, for optimal production of dextran, the most efficient medium will depend on the strain of organism being used." I n addition to the organisms using sucrose as a substrate, bacteria capable of synthesizing dextran from different carbohydrate sources have been discovered. Cultures of Acetobacter viscosum and Acetobacter capsulatum have been shown by Hehre and Hamilton'6 to convert amylodextrins to highly viscous products possessing serological properties like those of dextran. From this list of microorganisms capable of synthesizing dextran, it may readily be seen that one of the factors controlling the properties of the polysaccharide obtained is the strain of the particular bacterium used. This observation has been amply verified in a number of studies. Thus, immunological cross-reactions of dextrans with pneumococcus antisera,lBthe results of periodate oxidations,14J7and various physical measurements1*,lV have all demonstrated this point. When the dextran has been formed, it is usually isolated by the addition of ethanol to the culture filtrate.14*20 The precipitated dextran may be further purified by dissolving it in water and reprecipitating it with ethan01.14

IV. STRUCTURAL STUDIES The discussion of the structural aspects of the polysaccharide will be divided into two Sections. The f i s t deals with the chemical elucidation of the structure; the second, with the physical measurements made on dextran. A survey of the literature in this area has indicated that the fine structure of the dextran obtained is closely related to the particular strain that pro(14) A. Jeanes, w.C. Haynes, C. A. Wilham, J. C. Rankin, E. H. Melvin, M. J. Austin, J. E. Cluskey, B. E. Fisher, H. M. Tsuchiya and C. E. Rist, J . A m . Chem. Soc., 76, 5041 (1954). (15) E.J. Hehre and D. M. Hamilton, Proc. Soc. Ezptl. Biol. Med., 71,336 (1949). (16) J. Y.Sugg and E. J. Hehre, J . Immunol., 45, 119 (1942). (17) A. Jeanes and C. A. Wilham, J . A m . Chem. Soc., 72, 2655 (1950). (18) B. Ingelman and K. Siegbahn, Nature, 164, 237 (1944). (19) I. Levi, W.L. Hawkins and H. Hibbert, J . A m . Chem. Soc., 64, 1959 (1942). (20) A. Jeanes, W. C. Haynes and C. A: Wilham, J . Bucteriol., 71, 167 (1956).

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duces it.'J4 Furthermore, it appears that individual strains have the ability to mutate over the years, to the extent that the branches on their polysaccharides are, eventually, attached at points on the main chain different from those found originally.2' This type of behavior makes evaluation of the early structural studies difiicult. In view of this phenomenon, emphasis will be placed on structural determinations performed in the past few years. Most of the work with dextran has been conducted on material produced by various strains of Leuconostoc mesenteroides. For the sake of brevity in the followiiig discussion, the name of the organism will be omitted; it will be assumed that this common Genus was used, unless otherwise indicated. The B-512 and B-512 F designation is reserved for the particular substrain of Leuconostoc mesenteroides developed by the Northern Utilization Research Development Division of the U. S. Department of Agriculture, at Peoria, Illinois. 1. Chemical Studies

a. Methylation Studies.-The early methylation studies on dextran yielded a methylated dextran which, on methanolic hydrolysis, afforded a mixture of methylated ~-glueosides.~ From the fractional distillation of the resulting mixture, the methyl 2,3-di-O-methyl-, 2,3,4-tri-O-methyl-, and 2,3,4,6-tetra-O-methyl-~-glucopyranosides were ~ b t a i n e din , ~the ratio of 1:3 :1. This work was later criticizedz2on the grounds of incomplete methylation, inefficient distillation, and incomplete recovery. The dextran was subsequently re-inve~tigated,'~ in order to overcome these criticisms and to establish the structure of this particular dextran more definitely. Using improved techniques, Hibbert and coworkers were able to verify their earlier results,4 which have been substantiated by other workers.28 I n 1948, Stacey and Swiftz4characterized a new strain of Leuconostoc mesenteroides which they designated Belacoccus arabinosaceous (Birmingham). The dextran produced by this organism gave a ratio of methylated methyl aD-glucopyranosides similar to that reported previously by Hibbert and coworker^.^^ The physical properties of the Birmingham dextran were, however, different from those of the usual dextrans; this dextran could not, be converted into a water-insoluble gum by dehydrati0n.2~Four years later, a dextran produced by this same strain was shown, by methylation studies, 6) to contain (1 -+ 3) glucosidic linkages, in addition to the usual (1 (21) (1959). (22) (23) (24)

-

J. K. N. Jones and K. C. B. Wilkie, Can. J . Biochem. and Physiol., 37, 377

F. Brauns, Can. J . Research, B16, 73 (1938). W. Z. Hassid and H. A. Barker, J . Biol. Chem., 134, 163 (1954). M. Stacey and G . Swift, J. Chem. Soc., 1555 (1948).

345

DEXTRAN

Analysis of the methylated D-glucosides indicated a chain length of 6-7 D-glucose residues.26It was later demonstrated27that this same organism, cultured on a magnesium-deficient diet, produces a dextran with an average chain length of 40-50 units. The branch points were again shown, by methylation, to be of the (1 + 3) type, with no (1 + 4)-links being dete~ted.~T This observation gave support to the hypothesis that at least two enzymes are responsible for the synthesis of normal dextran. The enzyme responsible for the branching would, presumably, be magnesiumdependent. The structure of the B-512 dextran has been more thoroughly examined by methylation.28 The mixture of methylated sugars from the hydrolyzed, methylated polysaccharide afforded 2,4-di-O-methyl-, 2,3,4tri-0-methyl-, and 2,3,4,6-tetra-0-methyl-~-glucopyranose in the ratio of 1:21:1. This result established that B-512 dextran also contains the (1 + 3) glucosidic linkage at the branch point [instead of the earlier reported (1 + 4)-linkage].17 These findings indicated a range of structures for these highly dextrorotatory polysaccharides, defined by the following formula. -

1 L’6-(a-~-G)-1-I%-=

+ 64ff-D-Gl-l3

t 1 (wD-G) I 6

t 1 ( ~ - D I- G ) where G

=

glucopyranose.

Further studies28 have favored structures in which the side chain consists of only one D-glucose residue (x = 0). This structure will be discussed more thoroughly in the Section dealing with polymer studies (see p. 349). (25) S. A. Barker, E. J. Bourne, G. T. Bruce and M. Stacey, Chem. & I n d . (London), 1156 (1952). (26) S. A. Barker, E. J. Bourne, G. T. Bruce, W. B. Neely and M. Stacey, J . Chem. Soc., 2395 (1954). (27) S.A. Barker, E. J. Bourne, A. E. James, W. B. Neely and M. Stacey, J . Chem. Soc. 2096 (1955). (28) J. W. Van Cleve, W. C.Schaefer and C. E. Rist, J . Am. Chem. SOC.,78, 4435 (1956). (29) R. W. Jones, R. J. Dimler, A. Jeanes, C. A. Wilham and C. E. Rist, Abstracts Papers Am. Chem. SOC.,126, 1 3 (1954). ~

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W. BROCK NEELY

Jones and Wilkie,2l in a recent paper, describe a study of the structures of four dextrans currently being used as synthetic, blood-plasma expanders. These dextrans were shown to contain the (1 + 3)-linkage as the major branch point, with a small proportion of (1 + 4)-linkages present, in addition to the normal (1 4 6)-linkages. These conclusions21may, however, be attributable to incomplete methylation of the polysaccharides investigated. The dextran from the second species of Leuconostoc, namely, L. dextranicum, has received a less thorough investigation. In 1938, two accounts of a methylation study were reported.a0*81 Hibbert and coworkers80were unable to find any tetra-0-methyl-D-glucopyranosein the hydrolyzate of the methylated dextran. A closed-loop structure for this particular dextran was envisaged, to explain this observation.80 Peat, Schliichterer, and Stacey,81 however, were able to detect 0.23 % of the tetramethyl ether, and this finding indicated a chain length of approximately 500 D-glucose residues. In addition, they were able to isolate a mixture of di-0-methylD-glucopyranoses (lo%), a mixture which was not resolved. This mixture was later reported24 to contain mainly the 2,3-dimethyl ether, along with an unidentified dimethyl ether; this result is compatible with a structure containing other branch points, in addition to (1 4)-linkages. Daker and Staceya methylated the dextran produced by Betabacterium vermiformd (Ward-Mayer). Hydrolysis of the resulting material yielded a tri-0-methyl-D-glucopyr&nose and tetra-0-methyl-D-glucopyranosein a ratio of 25: 1; no dimethyl ether could be found, Osmotic-pressure studies indicated a chain of 500 D-glucose residues. The authors88 pointed out the striking similarity of this dextran to starch. Both polysaccharides have a molecular weight, as determined by physical measurements, which is a large multiple of that shown by end-group analysis. b. Periodate Oxidation.-The measurement of the amount of formic acid liberated in the periodate oxidation of a polysaccharide is a method for determining chain length that was first proposed in 1945." The method was later amplified, and improved for greater accuracy."J6 For critical surveys of this field, the reader may consult articles by Bobbitta7and Dyer.@ 182

-

(30) E. C. Fairhead, H. Hibbert and M. J. Hunter, Can. J . Research, BlB, 161 (1938). (31) S. Peat, E. Schluchterer and M. Stacey, Nature, 141,876 (1938). (32) 8.Peat, E. Schluchterer and M. Stacey, J . C h m . SOC.,681 (1939). (33) W. D. Daker and M. Stacey, J . Chem. Soc., 686 (1939). (34) F. Brown, S. Dunstan, T. G. Halsall, E. L. Himt and J. K. N. Jones, Nature, 166, 786 (1946). (36) T. G. Halsall, E. L. Hirst and J. K. N. Jones, J . Chem. SOC.,1427 (1947). (36) P. W. Kent, Science, 110,689 (1949). (37) J. M. Bobbitt, Advances in Carbohydrate Chem., 11, 1 (1966). (38) J. R. Dyer, in "Methods of Biochemical Analysis," D. Glick, ed., Interscience Publishers, Inc., New York, N. Y.,1966, Vol. 3, p. 111.

DEXTRAN

347

Jeanes and her coworkers were quite successful in utilizing a combined estimate of the production of formic acid and the consumption of periodate for classifying, into three groups,14*17*ae-42 the dextrans from many individual bacterial strains. The dextrans were arbitrarily assigned to their respective group depending on the percentages of (1 -+ 6), “(1 -+ 4)-like,” and ‘((1-+ 3)-like” linkages.ae The designations “(1 -+ 4)-like” and “(1 + 3)-like” follow from the fact that periodate uptake by, and formic acid liberation from, a dextran cannot supply information for differentiating between several different possibilities. The (1 4 4)-like linkages consisted of the (1 -+ 2)- or the (1 4 4)-linkage of the D-glucopyranose or the (1 --+ 5)linkage of the D-glucofuranose structure. The (1 + 3)-like linkage may be a combination of one or all of the following types: the (1 4 3)-, the (1 --+ 4)-, and the (1 + 2), or the (1 + 3)- and the (1 + 2) linkages of the D-glucopyranose residues. It should also be mentioned that the results of periodate oxidation do not permit one to distinguish between a (1 + 6)-linkage and the terminal, nonreducing residue of the polysaccharide. A study of the conditions involved in the periodate oxidation revealedasthat more reliable results are obtained if the iodine titrations are carried out a t 4’ instead of a t 25”. Such a technique provided data on the B-512 dextran that indicated 5 % of (1 4 3)-linkages [instead of (1 -+ 4)-linkages], as well as the usual 95% of (1 + 6)-linkage~.*~ This result was in agreement with those from previous methylation studies performed on the same material.” These intensive studies by this research group revealed that the dextrans show a wide variation in their fine structure, depending on the particular strain of bacteria used for the synthesis. Although the technique provides a rapid method for classifying the various dextrans, it affords no information concerning the exact nature of the linkages involved. An improved way of utilizing the method of periodate oxidation for determining the nature of the linkages in polysaccharides was provided by Smith and coworkers.M-46 They realized that, if the periodate-oxidized polysaccharide were reduced to the corresponding alcohol, the resulting fragments, on hydrolysis, would be indicative of the types of linkage present in the parent polymer. This scheme is shown in Fig. 1. Such an approach was used in a study of the dextrans from 6 different strains of L.mesente(39) J. C. Rankin and A. Jeanes, J. Am. Chem. Soc., 76,4435 (1954).

(40) A. Jeanes, W. C. Haynes and C. A. Wilham, J . Bacteriol., 7l,167 (1956). (41) C. A. Wilham, B. H. Alexander and A. Jeanes, Arch. Biochem. Biophys., 69, 61 (1956). (42) A. Jeanes, C. A. Wilham, H. M. Tsuchiya and W. C. Haynes, Arch. Biochem. Biophys. 71, 293 (1967). (43) M. Abdel-Akher, J. I(. Hamilton, R. Montgomery and F. Smith, J . Am. C b m . Soc., T4, 4970 (1962). (44) J. H. Hamilton and F. Smith, J . Am. Chem. Soc., 78,5910 (1966). (46) F. Smith and R. Montgomery, in Ref. 38, Vol. 3, p. 194.

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W. BROCK NEELY

r0ides.4~ The B-512 dextran, under these conditions, was shown to have 5 % of (1 4 3)-linkages; no evidence for (1 4 4)-linkages was obtained. This result once again confirmed the conclusion that this particular dextran contains (1 --+ 3)-linkages, in addition to the usual (1 + 6)-linkages. Neither of these methods, analysis of periodate consumption or isolation

FIQ.1.-Products Resulting from Periodate Oxidation, Reduction, and Hydrolysk of (1 -+ 6), (1 + 4), and (1 + 3) Linkages in Dextran.

of the organic products from the periodate oxidation, provides information about the position of the various residues in the polysaccharide molecule. Accordingly, the presence of an increased proportion of (1 4 4) - or (1 43)linked units should not, alone, be accepted as an indication of a higher degree of branching. It is known, for example, that at least two different linkages are present in the molecule (linear) of nigeran.q In addition, a high (46) J. W. Sloan, B. H. Alexander, R. L. Lohmar, Jr., I. A. Wolff and C. E. Rist., J . Am. Chem. SOC.,76, 4429 (1954). (47) S. A. Barker, E. J. Bourne and M. Stacey, J . Chem. SOC.,3084 (1953).

DEXTRAN

349

proportion of (1 ---f 6)-linked units, as indicated by the results of periodate oxidation, does not necessarily indicate that the molecule approaches linearity. A highly branched molecule has many nonreducing end-groups-which are indistinguishable from (1 .--) 6)-linked units.46 The continuing investigations by Smith and his coworkers (on controlled periodate oxidation followed by reduction and graded hydrolysis) will undoubtedly increase the importance of the periodate technique as a tool in ascertaining not only the type of linkages but also the location of the residues in the various polysaccharides.a-bO c. Acid Hydrolysis.-The partial, acid hydrolysis of dextran has resulted in the isolation and characterization of the disaccharide isomaltose as 6-0a-D-glucopyranosyl-D-glucopyranose. s1 3 2 Using enzymic hydrolysis of dextran, isomaltose and the next higher homolog, namely, isomaltotriose, have been obtained in 50 % and 20 % yield, respectively.ba The isolation of isomaltose in such a high yield furnishes excellent confirmation that the main structural linkage in the B-512 dextran is ~ r - ~ - ( l + 6). The partial hydrolysis of a highly branched dextran should produce di- and tri-saccharides containing linkages other than (1 + 6). Such evidence was found in the partially hydrolyzed mixture of dextran produced by Betacoccus arabinosaceous (Birmingham)26 A disaccharide behaving like nigerose (3-O-cr-~-glucopyranosyl-D-glucopyranose) and a trisaccharide whose properties were consistent with the presence of (1 -+ 6)- and (1+ 3)-linkages were found.26 This kind of evidence establishes the type of linkages that are present in the parent polymer, but it fails to establish the location of the residues. In order to ascertain the location of the linkages, a tetrasaccharide would have to be isolated and characterized. 2. Physical Studies a. Polymer Studies.-Early physical measurements on dextran (by electron microscopy,l8ultracentrifugation,'4 and birefringenceof flowb4) indicated that the polysaccharide exists as a branched, thread-like structure with a minimum thickness of 30-100 A and regions of swelling a t intervals of (48) J. K. Hamilton, G. W. Huffman and F. Smith, J . Am. Chem. Soc., 81, 2176 (1959); 81, 2173 (1959). (49) M. Abdel-Akher and F. Smith, J . Am. Chem. SOC.,81, 1718 (1959). (50) I. J. Goldstein, G. W. Hay, B . A. Lewis and F. Smith, Abstracts Papers Am. Chem. soc., 135, 3~ (1959). (51) M. L. Wolfrom, L. W. Georges and I. L. Miller, J . Am. Chem. Soc., 71, 125 (1949); L. W. Georges, I. L. Miller and M. L. Wolfrom, ibid., 69,473 (1947). (52) M. L. Wolfrom, A. Thompson and A. M. Brownstein, J . Am. Chem. Sac., 80, 2015 (1958). (53) A. Jeanes, C. A. Wilham, R. W. Jones, H. M. Tsuchiya and C. E. Rist, J . Am. Chem. SOC.,76, 5911 (1953). (54) A. Oronwall and B. Ingelman, Act& Physiol. Scand., 7 , 97 (1944).

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W. BROCK NEELY

about 800 A. Gronwall and IngelmanS4reported a molecular weight of several million, but accurate measurement was impossible because of inhomogeneity of the sample. A few years later, Jeanes and coworkers investigated the molecular association of dextran and related polysaccharides.66 By means of x-ray analysis, they were able to demonstrate an orderly molecular association in the B-512 dextran. Their results indicated that molecular associat:on decreases with an increase in the number of chains.66 The theoretical aspects of intrinsic viscosity and its relation to high polymers have been studied in detail.6' Viscosity may, in general, be regarded as a type of internal friction (in a solution) which opposes the relative motion of adjacent portions of the liquid. A knowledge of this property should provide information concerning the nature of the polymer in solution. An examination of the intrinsic viscosity of Swedish clinical dextran (a partially hydrolyzed dextran derived from a strain of Leueonostoc rnesenteroides) by Ingelman and Halling6?indicated that this particular dextran is very highly branched. Ogston and Woodsm re-examined Ingelman and Halling's data," taking into account the possibility of solvation, which is a measure of the effective volume occupied by the dextran in solution. Kuhn and Kuhn6' had previosly shown that the effect of branching, on a flexible chain, is to reduce the volume which the chain occupies in solution, compared to an unbranched chain of the same total length. Ogston and Woods were able to deduce that Ingelman and Halling's dextran was not only branched but was also highly so1vated.a These same authorsse were able to demonstrate similar characteristics for various samples of degraded dextran having an average molecular weight of 40,00&79,000. Wales, Marshall, and Weissberg'O made a theoretical study of intrinsic viscosity and its relation to the behavior of a branched polymer. They were able to calculate the ratio of the squares of the unperturbed radii of gyration of a branched to those of an unbanchal molecule (termed the g value). Such radii of gyration may be derived either from intrinsic viscosity data'O or from osmotic memurements61on dilute, dextran solutions. By varying the chain length and the spacing of the branch points on the main chain, they were (66)A. Jeanes, N.C. Shieltz and C. A. Wilham, J . BioZ. Chem., 178,617 (1948).

(66) See, for example: W. D. Lansing and E. 0. Kreemer, J . Am. Chem. Soc., W , 1369 (1936);H. A. Kraemers, J . Chem. Phys., 14, 416 (1946);M. L.Huggins, J . Am. Chem. SOC.,84, 2716 (1942);P. J. Flory and T. G . Fox, Jr., ibid., 78, 1904 (1961);W. Kuhn and H. Kuhn, Helv. Chim. A c h , 80,1233 (1947). (67) B. Ingelman and M. 8.Halling, Arkiv Kemi, 1,61 (1949). (68)A. G.Ogston and E. F. Woods, Nature, 171, 221 (1963). (69)A. G.Ogston and E. F. Woods, Truns. Faruduy Soc., 50,636 (1964). (60)M. Wales, P. A. Marahall and 8.G .Weissberg, J . Polymer Sci., 10,229 (1963). (61) E.Mariani, A. Ciferri and M. Maraghini, J . Polymer Sci., 18,303 (1966).

DEXTRAN

351

able to fit the observed data for B-512 dextran to a suitable model.s0 Branch lengths of 5-6 D-glucosyl residues, with a spacing of 9 units, were found to make the most satisfactory fit. It will be seen that this model is in fair agreement with the chemical determinations described earlier (see p. 345). These authorssoalso found that the presence of many short branches affects the intrinsic viscosity more than a few long chains. However, if the branches are close enough together to prevent free rotation about the bonds in the main chain, the effect of branching is lost and the molecule behaves as a linear chain.'O Senti and coworkerss2 made an exhaustive study (involving intrinsic viscosities, sedimentation constants, and molecular radii) on the B-512dextran ranging in molecular weight from 17,700 to 9.5million. They criticized the g-values of Wales and coworkers,soespecially those values at the higher molecular weights in the range of 324,000.The model based on the corrected g value would have branches 15 units long, spaced 5 units apart.62The chemical analysis of this particular dextran'?8s29indicated that 77 % of the branches were one unit in length; Senti and coworkerssawere therefore able to fit their observed g value to a model containing 77 % of the branches one unit long, with the remaining branches extending to lengths greater than 50. Thus, by the proper theoretical manipulation, the physical measurements on dextran can be made consistent with the structure deduced from chemical evidence. Further consideration led these authors to the conclusion that the long side-chains might be branched, in a manner equivalent to the main chain. This type af molecule fits the model for random branching deduced by Zimm and Stockmayer.6a In recent publications, Boveys'J'6 discusses this type of structure. His results were based on light-scattering measurements for the B-512dextran ranging in molecular weight from 50 million to 525 million. He assumed a linear, random coil, and calculated values for g based on reasonable estimates of the dimensions that such a model would give for dextran. In 2 0 such a model, he ran into diffiattempting to fit the chemical d a t a ~ * to culty. In order to have a model with the correct g value, the 20 % of long branches must be lo00 units long. However, as he pointed out,w for a model with branches this long, the random coil is impossible because of mutual interference of the branches. The easy way out of this apparent paradox would be to disregard the random-coil conformation and to assume a more rigid structure for the polymer in solution. (62) F.R. Senti, N. N. Hellman, N. H. Ludwig, G. E. Babcock, R. Tobin, C. A. Glass and B. L. Lamberts, J . Polymer Sci.,17, 527 (1965). (63) B. H. Zimm and W. H. Stockmayer, J . Chem. Phy8., 17, 1301 (1949). (64) F.A. Bovey, J. Polymer Sci.,86,169 (1959). (65) F.A. Bovey, J. Polymer Sci.,86, 183 (1959).

352

W. BROCK NEELY

Bovey, in the same series of papers, found that the molecular weight of the dextran continued to increase after the sucrose had all been utilized.M~66 This observation was explained on the basis that the branching enzyme continued to function and tacked preformed molecules to the main branch. Intrinsic-viscosity data helped to confirm this hypothesisp2,66 by demonstrating that the degree of branching increases as the molecular weight of the dextran increases. Fig. 2 shows a typical log-log plot of [q] against molecular weight.s2For molecular weights below 100,000, a linear relation holds. The curvature in the plot for larger molecular weights indicates a higher degree of branching. The dextran produced by Betacoccus arabinosaceous (Birmingham) has also been examined by means of intrinsic-viscosity data." These data in-

FIQ.2.-Log-log Dextran."

Plot of

[?I Against M,

for Fractions of Acid-degraded B-512

dicated that the clinical dextran from this organism was not as branched as the B-512 dextran. This is in direct conflict with the chemical evidence regarding the branching in the Birmingham dextran.26 There are at least two possible reasons for this discrepancy: (a) the partial hydrolysis (used in producing the clinical dextran) had stripped off most of the branches, or (b), as was pointed out previously,60the frictional effect of branching is lost and the molecule behaves like an unbranched chain, if the branches are closely spaced. b. Infrared Absorption Spectra.-Burket and Melvin" reported that dextrans from different organisms (in the Northern Utilization Research Development Division) showed different amounts of absorption at 794 em.-' (12.6~)in the infrared region of the spectrum. Other workers confirmed (66) L. H. Arond and H. P. Frank, J . Phys. Chem., 68,953 (1954). (67) G.C.Booth and V. Gold, J . Chem. SOC.,3380 (1956). (68) 8.C.Burket and E. H. Melvin, Science, 116,676 (1952).

353

D EXTRAN

this finding69J0and correlated the increased intensity at 794 cm.-' with the presence i n the dextran of periodate-resistant units; these were tcntatively regarded as being D-glucose residues linked (1 + 3), or (1 2) and (1 --f 4).89J0 Subsequently, this increased absorption was shown to be due to the presence of (1 + 3)-linkages?' The application of infrared analysis to some problems involved in structural studies on carbohydrates has been revie~ed.7~ Jeanes and her coworkers have utilized this characteristic absorption by (1 +3)-linkages in assaying the proportion of this linkage present in various d e x t r a ~ i s . lExcellent ~ * ~ ~ agreement was observed between the proportion of (1 -+ 3)-linkage as indicated by periodate oxidation and by infrared absorption. Other investigatorsz62 7 have employed infrared absorption spectra for predicting the presence, in dextrans, of various linkages which were later proved by methylation studies to be, indeed, present. It should be pointed out that, although infrared spectroscopy is a useful tool for the carbohydrate chemist, as with other physical methods, it does not supplant the classical chemical methods for determining the structure of polysaccharides. c. Optical Rotation.-Specific optical rotation is, in the field of carbohydrate chemistry, a useful physical constant and is readily obtained. Since optical activity is a function of the groups surrounding an asymmetric carbon atom, a change in optical activity is a reflection of a change in the structure of the molecule. Investigators at the National Bureau of Standa r d have ~ ~ examined ~ the specific rotation of clinical dextrans derived from a large assortment of organisms. They were unable to detect any significant differences in the optical rotations of these partially hydrolyzed dextrans. to +200° (in The values for [a] ranged slightly, but were very water, c 3-10 %). In the same investigation, no consistent differences in the refractive indices of the clinical dextran solutions were f0und.7~This consistency in the physical properties is undoubtedly a reflection of the similarity of the structures of the various dextrans examined. Jeanes and her collaborator^^^ were the first to make a systematic investigation of the specific rotations of the native dextrans derived from 96 different strains of bacteria. They obtained a rough correlation between [a]'i(in formamide) and the content of (1 + 3)-likelinkages present in the --f

':

(69) R. Lohmar, J . Am. Chem. SOC.,74, 4974 (1952). (70) A. Jeanes and C. A. Wilham, J . A m . Chem. SOC.,74, 5339 (1952). (71) S. A. Barker, E. J. Bourne, M. Stacey and D. H . Whiffen, Chem. & Ind. (London), 196 (1963); J. Chem. SOC.,171, 3468 (1954). (72) 9. A. Barker, E. J. Bourne and D. H. Whiffen, in Ref. 38, Vol. 3, p. 213. (73) W. B. Neely, Advances in Carbohydrate Chem., 12, 13 (1957). (74) C. F. Snyder, H. S. Isbell, M. R. Dryden and N. B . Holt, J. Research Natl. Bur. Standards, 53, 131 (1954).

354

W. BROCK NEELY

dextran. The analysis for (1 --+ 3)-linkages was carried out by using periodate oxidation and infrared ~pectroscopy~~ (see previous Sections for a discussion of these topics). Their ~orrelation'~ is reproduced in Fig. 3. One interesting observation made by these workers14 was that the content of (1 -+ 4)-like linkages does not appear to influence the specific rotation, 40

1149

a

0-

1355-9 @ll2l

*I142 1496-5. 142-5.

20

Qllll

.I191

i I

0 IWI-s

1396.

1374.

742.

@I139 81431

0 1433

81299-L -641 @I35I-L

I

I

235 230 [a125~ ( F O R M A M I D E ) .

I

240

1

245

FIG.3.-Correletion Between Specific Rotation and Content of (1 -+ 3)-like Link, rotation observed in formamide; @, rotation observed in ages in Dextran. (Key: . N potassium hydroxide and calculated to formamide by assuming that all dextrans show the same relative difference in rotation in these solvents14 as do dextrans B-512 and B-1355 8. The numbers designate different strains of micro-organisms in the Northern Utilization Research Development Laboratories a t Peoria, Illinois.)

whereas the specific rotation increases proportionally with the content of (1 + 3)-like linkages. This correlation has since been verified in aqueous solutions, where extrapolation to zero % of (1 + 3)-branches gave76 an [ a ] , of +194.5" for a hypothetical, linear dextran. Rowe reiterated the strong influence of the (1 + 3)-linkage on specific rotation?6J6 The characterization of dextrans by the optical rotation of their cupram(75) C. E. Rowe, Chem. & Ind. (London), 816 (1957). (76) Compare, for example, [CY]D +270° for nigeran [composed' of alternate -P 3) anda-D-(1+4) linkages] with [aln+200" for starch and for linear dextran. CY-D-(~

355

DEXTRAN

monium complexes has afforded some interesting results.77J8 lieeves79 has shown that cuprammonium-complex formation a t the 2,3-hydroxyl groups of D- lucopyranosides causes a large decrease in the optical rotation a t 4358 ,but complex-formation a t the 3,4-hydroxyl groups produces a large increase in optical rotation. Complexing at both the 2,3- and the 3,4-hydroxyl groups produces a relatively small shift. An examination of the rotations of the homologous series of the methyl ~ r - ~ - ( + l 6) poly-D-glucosides, ranging in degree of polymerization from 1-1 1, showed an increase in the negative shift in rotation of the cuprammonium complex.77 This observation was tentatively explained on the basis that small deviations ( ~6) poly-D-glucosides, from the “preferred” C1 formsoexist in the ( Y - D -+ and these deviations favor complexing at the 2,3-hydroxyl groups.77 By use of the value of -99,OOO” for the rotational shift of the (1 + 6)-linked unit, an equation was derived for resolving the (1 -+ 4)-like linkages (from periodate data) into (1 +4)- and (1 + 2)-linkages.?s By this method, large proportions of the (1 + 4)-like linkages in NRRL B-1299 dextran and in NRRL B-1399 dextran were foundls to be linked through position 2. This was the first time that a (1 + 2)-linkage had been reported for a native dextran. It is important that this result should be verified by methylation techniques. The use of cuprammonium rotational-shift data, in conjunction with other methods, should be of great assistance in the determination of the fine structure of polysaccharides.

1

V. ENZYMIC SYNTHESIS As stated earlier (see p. 342), study of the cell-free, enzymic synthesis of dextran was initiated by Hehre and Sugg6BBThey demonstrated the ability of a cell-free extract of Leuconostoc mesenkroides to catalyze the formation of dextran. The over-all reaction was represented by equation (I). In addin Sucrose

--f

(D-glucose),

+ n D-fructose

(1)

tion, Hehrgl was able to show that the reaction approaches well within 1 R of completion and is essentially nonreversible. This lack of reversibility might be explained by the rapid conversion of the liberated D-fructofuranose to the more stable fructopyranose form. The enzyme involved in this and it was soon synthesis was given the name dextransucrase by Hestrin,82v88 (77) T . A. Scott and F. R . Senti, J . A m . Chem. Soc., 77,3816 (1955). (78) T. A. Scott, N . N. Hellman and F. R. Senti, J . A m . Chem. SOC.,79,1178 (1957). (79) For a review, see R. E. Reeves, Advances i n Carbohydrate Chem., 6.107 (1951). See also, E. J. McDonald, J . Org. Chem., 26, 111 (1960). (80)This terminology is that introduced by R e e v e P to designate one of eight of

the many strainless form of the pyranoid ring. (81) E. J. Hehre, J . Biol. Chem., 168, 221 (1946). (82) 8. Hestrin, Nature, 164, 581 (1944). (83) S. Hestrin and S. Shapiro-Avineri, Biochem. J . , 38, 2 (1944).

356

W. BROCK NEELY

found to be a member of the general class of transglycosylases. Many reviews have been written on this subject and the reader is referred to relevant articles for a thorough discussion of these particular en~ymes.8*-*~ A few remarks may suffice to make more understandable the account following. Transglycosylases operate by transferring the glyoosyl group of the donor to a suitable acceptor. If the enzyme fails to discriminate against water as a n acceptor, the obvious end-result of the transferring reaction is simple hydrolysis. Such an enzyme is represented by invertase. For many years, the reaction of invertase on sucrose was thought to be a hydrolytic cleavage of the D-glucosidic (or D-fructosidic) bond. Bacon and coworkerss7-8@very clearly demonstrated, however, that a transfer mechanism is operating. In fact, they also demonstrated that other compounds containing a primary alcohol group can compete with water for the transferred moiety.89

It should be mentioned at the outset that dextransucrase is probably a mixture of at least two enzymes. This conclusion is based on the results of studies by Stacey and c ~ w o r k e r swho , ~ ~ demonstrated that a linear type of polymer is produced by a partially purified enzyme aged for two months at 0”.Further substantiation came from the discovery that dextran grown on a magnesium-deficient diet also shows a more linear structure.27 In addition, the recent work by Bovey, using light-scattering techniques, has demonstrated that a separate enzyme is responsible for most of the branching in the Bovey also presented evidence that the branching enzyme is more thermostable than the polymerizing enzyme.04The branching enzyme, as mentioned earlier (see p. 352), was believed to operate by transferring preformed chains to the main polymer.06This kind of reaction would not liberate any D-fructose, and, consequently, kinetic results based on liberation of D-fructose will be attributable to the polymerizing enzyme. In the following discussion, the term “dextransucrase” will be used for designating the enzyme responsible for the synthesis of the linear, CY-D(1 --+ 6) polymer of D-glucose. The ensuing presentation collates the various factors controlling the formation of the polysaccharide, and postulates a mechanism for the action of dextransucrase. The discussion will be based on equation (2) and will be divided into four parts: (1) acceptors, (2) substrate (donor), (3) enzyme, and (4)a mechanism.

I -

dextransucrase

+

Gp-(1 -+ 2) OFruj HOR Sucrose acceptor

G - OR products

+ FruOH

(84) S. A. Barker and E. J. Bourne, Quart. Revs. (London), 7,56 (1953).

(2)

(85) E. J. Hehre, Advances i n Enzymol., 11, 297 (1951). (86) J. Edelman, Advances i n Enzymol., 17, 189 (1956). (87) J. S. D. Bacon and J. Edelman, Arch. Biochem. Biophys., 28, 467 (1950). (88) J. S. D. Bacon, Biochem. J . , 67, 320 (1964). (89) H. J. Breuer and J. S. D. Bacon, Biochem. J.,66,462 (1957). (90) R W. Bailey, S A. Barker, E. J. Bourne and M. Stacey, J. Chem. SOC.,3530 (1957).

357

DEXTRAN

where G p = D-glucopyranosyl, Fruj an appropriate sugar-radical.

=

D-fructofuranosyl, and R =

1. Acceptors

Koepsell and his colleaguese1noted that the addition of certain sugars, notably maltose and isomaltose, to a dextran-synthesizing reaction-mixture causes the formation of short-chain oligosaccharides at the expense of highpolymer formation. This observation was most interesting, especially in the light of the ability to control the size of the polymer to an average molecular weight of 50,000 to 100,000 for “clinical” dextran. This study initiated the work since carried out by many investigators on the characteristics which endow a molecule with the ability to accept the transferred D-glucosyl group of sucrose.B2-108 The fact that the enzyme has not yet been obtained in pure form makes difficult an interpretation of the role of an added acceptor; and the identity of the natural acceptor-molecule has not yet been ascertained. Recently, however, an enzyme essentially free from any preformed dextran has been obtained.9e Further work along this line will make the work on acceptors easier to understand. An examination of Equation 2 indicates that, in the presence of an added acceptor, competitive reactions should be established between the natural and alternate acceptors. If the alternate acceptor is more efficieht than the natural acceptor, and if the activity of the added molecule is not impaired after successive transfers, a series of short-chain oligosaccharides will be the main products (91) H. J. Koepsell, H. M. Tsuchiya, N . N . Hellman, A. Karenko, C. A. Hoffman, E. 8.Sharpe and R. W. Jackson, J . BioE. Chem., 200,793 (1952). (92) R. W. Bailey, S. A. Barker, E. J. Bourne, and M. Stacey, Nature, 176, 1164

(1955). (93) 8. A. Barker, E. J. Bourne, P. M. Grant and M. Stacey, J . Chem. Soc., 601 (1958). (94) S. A. Barker, E. J. Bourne, P. M. Grant and M. Stacey, Nature, 178, 1221 (1956). (95) R. W. Bailey, S. A. Barker, E. J. Bourne, P. M. Grant and M. Stacey, J . Chem. SOC.,1895 (1958). (96)R. W. Bailey, S. A. Barker, E. J. Bourne, M. Stacey and 0. Theander, Nature, 179, 310 (1957). (97) W. B. Neely, Arch. Biochem. Biophya., 79, 164 (1959). (98) C. S. Stringer and H. M. Tsuchiya, J . Am. Chem. SOC.,80, 6620 (1958). (99) R. W. Bailey, S. A. Barker, E. J. Bourne and M. Staoey, J . Chem. SOC.,3536 (1957). (100) S. A. Barker, E. J. Bourne, P. M. Grant and M. Stacey, J . Chem. SOC.,601 (1958). (101) R. W. Jones, A. R. Jeanes, C. S. Stringer and H. M. Tsuchiya, J . Am. Chem. Soc., 78, 2499 (1956). (102) M. Killey, R. J. Dimler and J. E. Cluskey, J . Am. Chem. Soc., 77, 3315 (1955). (103) F. A. Bovey, J . Polymer Sci., 96, 191 (1959).

358

W. BROCK NEELY

of the enzymic reaction. If the added material is less efficient, competitive inhibition of the enzyme will occur. This logic indicates that, in order that a polymer of high molecular weight may be formed, the natural acceptor must either be present in very small concentrations or must be a very poor acceptor whose activity is increased tremendously as soon as the first transfer has taken place. There are two schools of thought on this question of primer action. The first group accept the hypothesis that preformed dextran, or some fragment thereof, is the initial acceptor; this theory is supported by evidence demonstrating that an enzyme preparation essentially free from any preformed dextran exhibits only slight activity when incubated in the presence of sucrose.ggThe second group (and the one to which the writer subscribes) favor the hypothesis that sucrose initiates its own polymerization. This theory is based on the following observations. (a) Dextransucrase preparations a t several stages of purification (and, consequently, containing different concentrations of carbohydrate and of dextran) exhibited similar activitie~.~8 (b) Linearity of rate as a function of enzyme concentrations8J04indicates absence of a diffusible ofa actor.^^^ ( c ) The addition of a solution of heat-inactivated enzyme to the reaction mixture did not cause an acceleration of rate.g8These facts support the conclusion that the natural acceptor is not to be found as a contaminant in the enzyme solution. There are several possible reasons for this difference of opinion. For example, the two schools used different strains of organisms for the production of the enzyme; this might conceivably account for the different results. In addition, Stacey and coworkers prepared their dextran-free enzyme by growing the organism in the presence of a large excess of maltose.geIn view of the author's worklo6on changing the specificity of the donor site of the enzyme with maltose, it is possible that the dextran-free enzyme isolated by Stacey and coworkersgsmight have properties very different from those of the natural enzyme. On the assumption that the enzyme solution is not contaminated with an acceptor, the tentative conclusion is reached that sucrose must be capable of acting as its own acceptor. Several factors give credence to this idea. A trisaccharide I was recently isolated and charact e r i ~ e d . ~ ~The J ~ 7addition of I to the enzyme-sucrose mixture caused a marked increase in the liberation of D-frUctose.gKTrisaccharide I might be the initial oligosaccharide formed by the transfer of a D-glucosyl group to sucrose. It is, presumably, a more efficient acceptor than sucrose; conse(104) W. B. Neely, J . A m . Chem. SOC.,80, 2010 (1958). (105) J. 9. Friedenwald and G. D. Maengwyn-Daviea, in Symposium on Mechanism Enzyme Action, Johns Hopkins Univ. McCollum-Pratt Znst. Contrib., 70, 173 (1954). (106) W. B. Neely, J . A m . Chem., Soc., 81, 4416 (1959). (107) 9. A. Barker, E. J. Bourne and 0. Theander, J . Chem. SOC.,2064 (1957).

DEXTFLAN

359

quently, the reaction leads, eventually, to the formation of a product of high molecular weight (assuming that further transfers to I do not impair its ability to accept D-glucosyl groups). Hehrell established the presence of a 8-D-fructofuranosyl group at the reducing end of a dextran chain; the dextran was a polymer of low molecular weight produced by Streptococcus DS strain 50. This result indicates that sucrose is capable of serving as its own chain-initiator. The fact that sucrose associates with the acceptor site was shown from kinetic evidence.98JOaJOs The data indicated a n inhibition of dextransucrase at high concentrations of sucrose; this result was amenable to interpretation on the basis of a mechanism involving true inhibition by the substrateg8Josrather than to a lowered concentration of water.s1 Such a lowering of activity in the presence of excess sucrose was explained on the basis of complexing with the acceptor sitegsJ@;this implies that, although sucrose is capable of being bound, it is not very efficient in the role of acceptor. However, when once the transfer of a D-glucosyl group has taken place, forming I, chain propagation becomes very rapid.gaThese characteristics of sucrose are precisely those which would be expected for the production of a polysaccharide of high molecular weight, namely, an acceptor of very low binding order, whose activity is greatly enhanced when once the initial transfer has taken place. The results of work on the various acceptors for this enzyme permit a few speculations to be made concerning certain structural requirements which appear to be necessary. The most efficient acceptors thus far encountered are isomaltose, maltose, methyl a-D-glucopyranoside, and D-ghC O S ~ I. n~ every ~ case, the transferred D-glucosyl group enters at position 6 of the nonreducing moiety of the acceptor molecule; this indicates that a D-glucopyranose moiety is one factor that is important. The fact that ~-galactose,~l lactose,Ss and raffinoseS7 are not so efficient, and that D-mannoseg1is inert, substantiated this conclusion. This brings out the interesting observation on points of attachment of the acceptor to the enzyme. D-Mannose and D-galactose differ from D-glucose in that, in D-mannose, the hydroxyl group a t C2 is L (instead of D), and, in D-galactose, the hydroxyl group at C4 is L (instead of D). It seems reasonable to suppose that correct orientation of these particular hydroxyl groups is necessary in order that (108) W. B. Neely, Nature, 182, 1007 (1958).

360

W. BROCK NEELY

attachment may occur. The ability of 3-O-methyl-a-~-glucopyranose to act as an acceptorgsrules out the C3-hydroxyl group as the point of attachment. From this discussion, it follows that a compound containing the D-XY~O configuration but lacking the 5-(hydroxymethyl) group should be an excellent inhibitor for this enzyme. Such a compound will be accommodated on the acceptor site and should be quite unreactive as far as chain initiation is concerned. This structure is possessed by methyl a-D-xylopyranoside,and preliminary experiments indicate that this glycoside is a competitive inhibitor of the dextransucrase system.10e Certain disaccharides possessing a D-glucopyranosyl group as the nonreducing moiety do not show high abilities as acceptors. These include cellobiose,96 l e u c r o ~ e sucrose,e1 ,~~ and trehaloseel; indeed, the last-named sugar is reported to be inert in this reacti0n.~1The explanation for this apparent anomaly will have to await further knowledge concerning the conformations of oligosaccharides. For example, the different behavior of cellobiose and maltose might be related to the difference in their stable conformation, as described by Reeves1l0and Bentley.I1l One further point may be considered. Cellobiose, lactose, and rafhose have one feature in common. In the synthesis under discussion, the resulting oligosaccharide, formed by a transfer of a D-glucosyl group, is branched. The branching occurs at C2 of the reducing moiety in cellobioseQ6and lact0se,~6and at C2 of the D-glucose residue of raf€in0se.~7A comparison of the structure of raffinose with that of I illustrates again the important part played by the orientation of the C4-hydroxyl group of the terminal glycose unit. With rafiose and lactose, the D-galacto configuration of the terminal unit causes the transferred group to enter at the position previously described. It will be interesting to examine the series of oligosaccharides formed when melibiose and D-galactose are used as alternate acceptors. It is just possible that the resulting oligosaccharides will also show the branched type of structure. Fig. 4 depicts the molecules of maltose and lactose. Assuming that the C2- and C4-hydroxyl groups of the nonreducing moiety of maltose fit the acceptor site as shown in Fig. 4, it becomes a matter of interest to see how some of the other acceptors fit this pattern. There is only one way in which lactose can be so oriented that two hydroxyl groups occupy locations similar to those of the C2- and C4-hydroxyl groups of the D-glucosyl residue of maltose; this orientation is shown in Fig. 4. It will also be noticed that this arrangement of lactose places the C2-hydroxyl group of the D-glucose residue in approximately the same position as the primary hydroxyl group of (109)W.B. Neely, Unpublished results. (110) R. E. Reeves. J , Am. Chem. SOC.,76,4595 (1954). (111) R. Bentley, J . Am. Chem. Soc., 81,1952 (1959).

DEXTRAN

361

maltose, and these are precisely the positions at which the transferred D-glucosyl group enters the respective acceptors. 2. Substrate One of the main characteristics of this enzyme system is the unique role played by sucrose as the donor of D-glucosyl groups. Even the closely allied isomer, a-D-galactopyranosyl 0-D-fructofuranoside is inactive in this enzymic reaction.l12 The results of work on the enzymic synthesis of starch from D-glucosyl phosphate raised a question as to whether a phosphoryla-

FIG.4.-Models of Lactose and Maltose. (See the text for a discussion.)

tion step might be involved in dextran formation. However, it was unequivocally demonstrated that dextransucrase does not require a phosphorylated sugar in the synthesis.113 Energy-wise, sucrose is well equipped to play its role as donor. The presence of the D-fructofuranoside moiety endows sucrose with a very labile glycosidic bond,l14making the formation of dextran thermodynamically possible. The high selectivity of the enzyme for sucrose indicates, also, that the enzyme surface has a geometry which allows no other sugar to make contact with the donor site. Recently, it has been shown that dextransucrase is very labile to heat (non-reversible de(112) D.S.Feingold, G. Avigad and S. Hestrin, J . Biol. Chem., 224, 295 (1957). (113) E.J. Hehre, Proc. SOC.Ezptl. Biol. Med., 64, 240 (1943). (114) W.Z.Hassid and C. E. Ballou, in “The Carbohydrates: Chemistry, Biochem-

istry, Physiology,” W. Pigman, ed., Academic Press Inc., New York, N. Y., 1957, p. 489.

400 300

zoo KM. I00

80 60 40 30 20

I

10

3.24

I 3.29

I 3.34

I I 3.39 3.44 IIT x lo3.

I 3.49

I

3.54

3.59

FIG.5.-A Semi-log Plot of the Michaelis Constant Against the Reciprocal of the Absolute Temperature.106 0.8

0.6 0.4 0.3

0.2

0.04 0.03

3.24

3.29

3.34

3.39

I/T x

3.44

3.49

3.54

3.59

103.

FIG.6.-A Semi-log Plot of the Maximum, Initial Velocity Against the Reciprocal of the Absolute Temperature.lo6 362

DEXTRAN

363

naturation above 35") and to urea denaturation.lo6 A plot of Michaelis constant against the reciprocal of the absolute temperature (given in Fig. 5) shows some interesting facts. Above 30°, there is a marked increase in the Michaelis constant; this indicates a greatly reduced affinity of the enzyme for the substrate. However, as Fig. 6 illustrates, when once contact has been made, the enzyme-substrate complex decomposes to afford the product, in a fashion predicted by the Arrhenius equation. The explanation for this observation rests on reversible denaturation, wherein the enzyme becomes partly unfolded and thus loses some of its specificity for sucrose.1o6 While it is in this partly unfolded state, the enzyme is capable of transferring a D-glucosyl group from maltose to maltose, with the formation of a trisaccharide and the liberation of D-glucose.'06 The reaction is not particularly extensive, but this can be explained by the low energy of the maltosidic linkage (as contrasted with the energy available in sucrose). Although sucrose is the most efficient, donor molecule thus far encountered, the above work indicates that the specificity pattern of the enzyme can be a1t ered. 3. Enzyme Koshland, Ray, and Erwinlls have proposed an attractive theory for the structure of the enzyme. These authors differentiate between the catalytic site and the specificity site of the enzyme. They were able to demonstrate that a rearrangement of the amino acids concerned with the specificity of phosphoglucomutase endows this particular enzyme with proteolytic activity. The results reported on using maltose as a substrate for dextransucrase106lend support to this attractive theory.lls Further purification of dextransucrase will be necessary before any exact knowledge concerning the specificity site can be gleaned. On the other hand, the catalytic site, by its very nature, lends itself to a kinetic study. Using this approach, certain characteristics regarding the catalytic site have been uncovered. The influence of hydrogen ions on the catalysis has been investigated.lo4From the nature of the bell-shaped activity curve, resulting from a plot of activity against pH, several deductions were made.10* The pK's of the functional groups on the catalytic site corresponded with those of a carboxyl group and an imidazole group. The implication that an imidazole group is a part of the catalytic site was later substantiated116by application of the photooxidation technique for destroying the imidazole group in proteins.ll7 The discovery of the presence of an imidazole group at the catalytic site of this (115) D.E.Koshland, Jr., W. J. Ray, Jr., and M. J. Erwin, Federation Proc., 17, 1145 (1958). (116) W. B. Neely, Arch. Biochem. Biophys., 79, 297 (1959). (117) L. Weil, A. R. Buchert and J. Maher, Arch. Biochem. Biophys., 40, 245 (1952).

364

W. BROCK NEELY

enzyme is most interesting, since it gives support to the hypothesis advanced by Koshland and coworkers116that the transferring enzymes all have in common a definite sequence of amino acids. Kinetic evidences8J18 indicated, also, that a simultaneous binding of the acceptor and donor must take place before polymerization occurs. Binding of the donor (sucrose) on the acceptor site causes inhibitione8Jm;and excess acceptor, in the presence of a low concentration of sucrose, also causes inhibitions8 (because of competitive interaction of the acceptor and sucrose for the donor site). Any mechanism that is postulated for this reaction must, therefore, include a three-component complex-between the enzyme and both substrates. 4. Mechanism of Polymerization

Polymerizing reactions are of two basic types: (a) the polycondensation reaction, characterized by a well-defined, unit step (such as esterification), and (b) the addition- or chain-reaction, represented by the vinyl, or diene, class of compounds. The chain reaction may be further classified into that explicable on the single-chain hypothesis and that for which the multichain hypothesis has been a d v a n ~ e dThe . ~ ~polymerization ~~~ of carbohydrates is a condensation, since the elements of water are eliminated when two glycose molecules are joined together. However, the process differs from “condensation polymerization,” because short-chain oligosaccharides do not react with other short chains to give molecules having the wide distribution of molecular weights which is so characteristic of this type of polymerization. The formation of polysaccharides, and of dextran in particular, appears to follow the mechanism for chain polymerization, in that three well-defined steps may be distinguished : initiation, propagation, and termination.11sThe question then arises as to whether the dextran synthesis is a single-chainor a multi-chain reaction.s6In other words, does the enzyme build a complete molecule of dextran before starting synthesis of the next one, or are the chains built up in a number of discrete (that is, discontinuous) steps? Stacey,120 in 1943, tentatively suggested that the enzyme remains in combination with the growing chain. This suggestion implies that the single-chainmechanism operates; the molecular weight would be high in the initial phase of the reaction and would remain approximately constant. This suggestion has been partially verified by a failure to detect any oligosaccharides in the early stages of the dextran ~ynthesis.~’ The results of (118) W. B. Neely and C. F. Thompson, Nature, 184, 64 (1959). (119) H. Markand and A. V. Tobolsky, “Physical Chemistry of High Polymer Systems,” Interscience Publishers, Inc., New York, N. Y., 2nd Edition, 1950. (120) M. Stacey, Chem. & Ind. (London), 62, 110 (1943).

DEXTRAN

365

molecular-weight d e t e r m i n a t i o n ~ ~ ~ have J ~ ' also tended to establish this mechanism. B o ~ e yin, ~addition ~ to showing (by light-scattering measurements) that the molecular weight of the polymer is very high (50 X 106) in the early stages of the reaction, found that the molecular weight continues to increase after all of the sucrose has been utilized. This finding was explained on the hypothesis of the presence of a branching enzyme, in addition to the polymerizing enzyme. The theory was verified by showing that the chain-initiating enzyme is partly destroyed at 35", but that the branching enzyme continues to function.64Bovey 66 elaborated on his observations (see Section 111, 2a) and made the assumption that the polymerizing enzyme causes the transfer of one D-glucosyl residue to the C3 position of the backbone. The branching enzyme then accounted for the other 20 % of the observed (1 3)-linkages by attaching additional chains to the main chain. This would endow the branching process with the characteristics of a polymerization of the true condensation type. The course of the enzymic reaction in the presence of added acceptors J~~ presented a appears to follow a multi-chain r e a c t i ~ n . ~ ~ Weibu11lZ2 statistical analysis of previous worklo'; this analysis indicated that, when methyl a-D-glucopyranoside is the added acceptor, the synthesis proceeds by way of a polymerization of the ethylene oxide type or by a true multichain reaction. BoveylOS has verified this reaction scheme and has also demonstrated the existence of a competing type of reaction, wherein the added acceptor (methyl a-D-glucopyranoside) reduces the concentration of the polymer of high molecular weight without affecting its molecular weight. I n other words, the added acceptor gives rise to a second D-fructose-producing reaction (from sucrose) which competes with the natural synthesis of dextran of high molecular weight. Thus, dextran synthesis presumably occurs as follows. In the absence of an added acceptor, the polymerization is initiated (very inefficiently) by sucrose molecules. However, once initiated, the propagation proceeds very rapidly, since the enzyme remains with the growing polymer chain, and the activation energy required for the transfer is not lost after each step. The reaction is terminated by dissociation of the enzyme from the polymer. On the other hand, the alternative acceptor molecules initiate polymer formation very efficiently; propagation proceeds slowly, and the molecular weight gradually increases, in a true multi-chain reaction. The following mechanism is a slight modification of the scheme proposed by Boveylo3for explaining the enzymic synthesis of dextran. The step con--f

(121) H. M. Tsuchiya, N. N. Hellman, H. J. Koepsell, J. Corman, C. s. Stringer, S. P. Rogovin, M. 0. Bogard, G. Bryant, V. H. Feger, C. A. Hoffman, F. R . Senti and R . W. Jackson, J . A m . Chem. Soc., 77, 2412 (1955). (122) B. Weibull, Acta. Chem. Scand., 12, 568 (1958).

366

W. BROCK NEELY

trolled by kffz represents inhibition of the enzyme in the presence of excess sucrose. The inhibition supposedly occurs by an absorption of the D-fructose moiety of sucrose onto the receptor site of the enzyme, as contrasted with the absorption of the D-glucose moiety (step kz), which normally occurs. This mechanism is strictly a “pictorial” description of this particular inhibition.

E:

= the enzyme, with a donor and a receptor site,

G = the D-glu-

copyranosyl moiety of sucrose, and F = the D-fructofuranosyl moiety of sucrose, A = the alternative acceptor, k2 = the absorption step, ka = the initiation step, ka = the propagation step (fast), lcr, = the termination step, k’a = the slow, multi-chain step (GA, in turn, combines with enzyme), kNz = substrate inhibition, and kf2 = the efficient binding of acceptor (k’z > k2).

VI.

USES

The dextrans have been proposed for use in a number of industrial and medical applications. The greatest single use has, however, been in the latter field, where partially hydrolyzed dextrans have been used as substi-

367

DEXTRAN

TABLEI Patents Issued on Dextran for Various Applications Patent No.

Application

U. 9. 2,734,066 U. S. 2,706,690 U. S. 2,734,055 U. S. 2,734,828 U. S. 2,749,277 U. S. 2,756,156 U. S. 2,756,160

Setting twist in yarn Coating cotton yarn (alkyl ether) Water-resistant textiles (ester) Lacquer (ester and ether) Coating on body powder Rustproofing composition (ester/oil) Finishing textiles (like the use of starch) Seed coating Coating regenerated cellulose articles (xanthate) Bonding fibers (xanthate) Adhesive Bonding collagen strands Adhesive Preserving shrimp Preserving shrimp Filament (ether) Anticoagulant (sulfate)

U. S. 2,764,843 U. S. 2,768,097 U. U. U. U. U. U. U. U.

S. 2,736,652 S. 2,746,880 S.2,748,774 S. 2,768,096 S. 2,758,929 S.2,758,930 S.2,702,231 S. 2,715,091

U. U. U. U. U. U. U. U.

S. 2,716,084 S. 2,716,237 S. 2,742,399 S. 2,725,303 S.2,731,349 S. 2,731,015 S. 2,733,326 S. 2,736,710

U. S. 2,409,816

Dextranase Dextranase Dextranase Pigmented dextran Edible container Artificial sponge (foam) Azo dyes Varnish remover (with isopropyl alcohol) Stabilizer for barium sulfate suspension (carboxymethyl-) Lipstick base (ester) Soil stabilization (syn. in soil) Prolonging foam life Wood stain (azo dye) Cigarette filter tips Resinous coatings (with urea-formaldehyde) Filament (with urea-formaldehyde) Battery separator plates (with alkyd resin) High-viscosity sirup

U. S. 2,360,327

Drilling-muds

U. S. 2,746,906 U. S. 2,749,276 U. S. 2,756,134 U. 8. 2,768,926 U. S. 2,762,679 U. S. 2,768,913 U. S.2,674,584

U. S. 2,674,517 U. S. 2,624,768

Assigned to

Commonwealtha Commonwealth Commonwealth Commonwealth Commonwealth Commonwealth Commonwealth Commonwealth Commonwealth Commonwealth Commonwealth Commonwealth Commonwealth Commonwealth Commonwealth Commonwealth Nat’l. Res. Dev. Corp., London Commonwealth Commonwealth USA (Dept. of Agr.) Commonwealth Commonwealth Commonwealth Commonwealth Commonwealth Midland Chemical Corporation, Dayton Commonwealth Commonwealth Commonwealth Commonwealth Commonwealth Commonwealth Commonwealth Commonwealth (D. V. Wadsworth and M. F. Hughes) (L. H. Bailey, W. L. Owen, and W. L. Owen, Jr .)

368

W. BROCK NEELY

TABLE1-Continued Pal& No.

A pplkation

U. S. 2,587,623 U. S. 2,602,082

Acylation of dextran in formamide High-viscosity dextran by reaction with aldehyde Reaction of carbohydrate with quaternary ammonium hydroxide Dextran acetate Ester derivative of dextran Dextran ether esters Mixed benzyl and butyl ethers

USA (Dept. of Agr.)

Dextran base wrinkle-drying compositions

New Wrinkle, Inc.

Dextran acetate Dextran benzyl ether Dextran benzyl ether Acetone-soluble benzyldextran Dextran ether Benzyl ether of dextran Lacquer with benzyl ether of dextran -etherify or esterify in meDextran dium benzyl -soft, rubbery ether ether -from separated dextran -dehydrated, acetone-solubl Dextran dentifrice preparations Moistureproof cellophane sheets (Carboxymet hyl) dext ran as binder for tobacco particles Dextran benayl ethers Ether of polysaccharide gum Benzyl ether Dextran ethers Use in beverage Anticoagulant (sulfate)

Chemical Dev. Corp. Chemical Dev. Corp. Chemical Dev. Corp. Commonwealth Chemical Dev. Corp. Chemical Dev. Corp. Commonwealth

U. S. 2,663,526 U. S. 2,344,190 U. S. 2,229,941 U. S. 2,239,980 U. S. 2,249,544 U. S. 2,503,622 U. S. 2,503,623 U. S. 2,503,624) U. S. 2,386,994 U. S. 2,385,553 U. S. 2,380,879 U. S. 2,328,036 U. S. 2,344,179 U. S. 2,344,180 U. S. 2,236,386 U. S. 2,203,702 U. S. 2,203,703 U. S. 2,203,704 U. S. 2,203,705

1

U. 8.2,779,708 U, S. 2,766,143 U. S. 2,778,753 Can. 394,662 Can. 391,240 Br. 517,820 Br. 517,397 Swiss 217,217 Swed. 118,014 n

Commonwe

1

Assigned to

Keever Starch Co. Chemical Dev. Corp. Commonwealth Commonwealth Commonwealth

Commonwealth Colgate-Palmolive Commonwealth Commonwealth Commonwealth Commonwealth Stahly and Carlson Martens

;h: Commonwealth Engineering Company, Dayton, Ohio.

tutes for blood plasma in the treatment of shock.12a This single application provided the impetus for initiating much of the work that has been reported in this Chapter, as well as for numerous investigations on the pharmacology of “clinical” dextran. Only a few of the many reports in this (123) B. Ingelman, Acta Chem. Scand., 1, 731 (1947). (124) C. R. Ricketts, L. Lorena, W. d’A. Maycock, Nature, 166, 770 (1950).

DEXTRAN

369

area will be mentioned. For a complete bibliography, the reader may con,lZ6 sult two excellent articles.126 For use as a blood-plasma substitute, the dextran should have a molecular weight in the range of 50,000 to 100,000. This criterion has occasioned a concerted effort to produce dextrans in the correct range. Partial, acid hydrolysis of native dextran followed by fractionation with various solvents, or enzymic production of dextran of low molecular weightlZ7are methods which have been used. In addition, ultrasonics has been suggested as a means of depolymerizating native dextran to the correct size for clinical use.12*s129 Introduction of alternative acceptors into the reaction mixture for enzymic synthesis has also been employed for this purposeg1;it is described in an earlier Section of this Chapter. Attempts have been made to use dextran and various derivatives in many additional ways; these include the use of sulfated dextran as a synthetic analog of heparin130Ja1 and of dextran in oil-well drilling muds.la2 Table I presents a comprehensive list of the application patents that have been issued on dextran. (125) W. L. Chessman, “Blood and Blood Plasma Subst#itutes,” U.S. Dept. Agr., Bur. Agr. and Ind. Chem., Mimeo. Circ. Ser. AIC 289, 177 (1950). (126) J. M. Howard, R. V. Ebert, W. L. Bloom and M. H. Sloan, A m . J . Surgery, 97, 593 (1959). (127) N. N. Hellman, H. M. Tsuchiya, S. P. Rogovin, R. W. Jackson and F. R. Senti, U. S. Pat. 2,726,985 (1955). (128) M. Stacey, Research (London), 4, 48 (1951). (129) V. C. Haworth, British Pat. 681,548 (1952). (130) C. R. Ricketts and K. W. Walton, Chem. & Znd. (London), 1062 (1951). (131) C. R . Ricketts, Biochem. J . , 61, 120 (1952). (132) W. L. Owen, Sugar, 46, No. 7,28 (1951).