NATURAL SYNTHESIS OF LOW MOLECULAR WEIGHT (CLINICAL TYPE) DEXTRAN BY A STREPTOCOCCUS STRAIN* BY EDWARD J. HEHRE (From the Department of Microbiology and Immunology, Cornell University Medical College, New York, New York) WITH A NOTE BY FREDERIC R. SENTI AND NISON N. HELLMAN (Received for publication, February 14, 1956) The present paper describes a novel bacterial dextran’ of a molecular weight lower than that of the native dextrans hitherto obtained as major products from Leuconostoc mesenteroides or other species. As with several dextrans of unusual origin reported earlier (2, 3), this example was first recognized by the use of immunological reactions capable of distinguishing dextrans of different types and sizes from one another, and from other glucose polymers such as starches and glycogens. In 1946, Hehre and Neil1 showed that certain non-hemolytic streptococci (Streptococcus var. DS) from the blood of patients with subacute endocardi- tis, and found also in the throats of healthy persons, convert sucrose to products serologically identifiable as dextrans (2). Subsequent examina- tion of more than 100 of these DS streptococci revealed that the sucrose broth culture fluids of one particular strain (No. 50) behaved more like solutions of partly hydrolyzed than of native dextran as judged by tests for precipitation by type 2 and type 20 pneumococcus rabbit antiserums (4, 5) and by precipitation with alcohol. The present data establish that the sucrose-derived product of Streptococcus 50 is in fact a predominantly 1 ,6-linked a-anhydroglucose polymer, the greater part of which is similar in molecular size to the plasma substitutes made from partly hydrolyzed leuconostoc dextrans of extremely high molecular weight. These findings indicate that the product might be a highly advantageous starting material for the production of clinical dextran. Elimination of the hydrolysis step would simplify the current preparative process and possibly also increase its efficiency. Evidence has been obtained that the small size of the streptococcal dex- tran molecules is the result of synthesis by an uncommon type of dextran- sucrase. Hitherto, inherent catalyst specificity had not been counted * This work was supported by a grant from the Corn IndustriesResearch Founda- tion. 1 A preliminary report was presented at the Fifty-second national meeting, Society of American Bacteriologists, Boston, Massachusetts, 1952 (1). 739 by guest on March 25, 2020 http://www.jbc.org/ Downloaded from
Transcript
NATURAL SYNTHESIS OF LOW MOLECULAR WEIGHT (CLINICAL TYPE) DEXTRAN BY A
STREPTOCOCCUS STRAIN*
BY EDWARD J. HEHRE (From the Department of Microbiology and Immunology, Cornell University
Medical College, New York, New York)
WITH A NOTE BY FREDERIC R. SENTI AND NISON N. HELLMAN
(Received for publication, February 14, 1956)
The present paper describes a novel bacterial dextran’ of a molecular weight lower than that of the native dextrans hitherto obtained as major products from Leuconostoc mesenteroides or other species. As with several dextrans of unusual origin reported earlier (2, 3), this example was first recognized by the use of immunological reactions capable of distinguishing dextrans of different types and sizes from one another, and from other glucose polymers such as starches and glycogens.
In 1946, Hehre and Neil1 showed that certain non-hemolytic streptococci (Streptococcus var. DS) from the blood of patients with subacute endocardi- tis, and found also in the throats of healthy persons, convert sucrose to products serologically identifiable as dextrans (2). Subsequent examina- tion of more than 100 of these DS streptococci revealed that the sucrose broth culture fluids of one particular strain (No. 50) behaved more like solutions of partly hydrolyzed than of native dextran as judged by tests for precipitation by type 2 and type 20 pneumococcus rabbit antiserums (4, 5) and by precipitation with alcohol. The present data establish that the sucrose-derived product of Streptococcus 50 is in fact a predominantly 1 ,6-linked a-anhydroglucose polymer, the greater part of which is similar in molecular size to the plasma substitutes made from partly hydrolyzed leuconostoc dextrans of extremely high molecular weight. These findings indicate that the product might be a highly advantageous starting material for the production of clinical dextran. Elimination of the hydrolysis step would simplify the current preparative process and possibly also increase its efficiency.
Evidence has been obtained that the small size of the streptococcal dex- tran molecules is the result of synthesis by an uncommon type of dextran- sucrase. Hitherto, inherent catalyst specificity had not been counted
* This work was supported by a grant from the Corn Industries Research Founda- tion.
1 A preliminary report was presented at the Fifty-second national meeting, Society of American Bacteriologists, Boston, Massachusetts, 1952 (1).
among the factors (6-8) that influence or determine the molecular weight of the polymer product in dextran synthesis.
EXPERIMENTAL
Native Soluble Dextran of Streptococcus DS, Strain 60
Stock cultures of Streptococcus 50 were maintained in meat infusion broth containing 5 per cent sterile defibrinated rabbit blood, or in broth comprising 1 per cent yeast extract, 1 per cent tryptose, 0.3 per cent NaCl, 0.2 per cent K2HP04, and 0.03 per cent glucose. For the production of dextran, media containing 20 per cent sucrose were used. Cultures grown with lower concentrations of sucrose, and under various conditions of temperature, aeration, and time, however, all yielded a product similar to the following.
Preparation 18S-3 liters of broth which contained 20 per cent sucrose, 1 per cent tryptose, 0.5 per cent Difco yeast extract, and 0.75 per cent KzHPOll were inoculated with 60 ml. of a 17 hour, 5 per cent sucrose broth culture of Streptococcus 50. After incubation at 37” for 3 days without agitation, the culture, which had reached pH 4.5 but was not notably viscous, was brought to pH 7.0 with NaOH and centrifuged. Ethanol was added to the supernatant fluid to a concentration of 65 per cent by volume, the mixture was held at 25” for 2 hours, and the voluminous pre- cipitate was collected by centrifugation. Most of the crude dextran dis- solved readily on addition of 2 liters of distilled water at 25” and, after re- moval of an insoluble fraction (dry weight 4.75 gm.), was reprecipitated by ethanol at 65 per cent concentration. The product was triturated with ethanol and dried in vacua at 25’ over CaCl*; it weighed 117 gm. on the moisture-free basis, a yield of 41 per cent of the theoretical maximum. Some of the properties of this soluble native material, 18S, are given below.
Precipitability by Methanol-Direct comparison was made with the native dextrans produced by three other bacteria and with several partly hydrolyzed and fractionated dextrans used as plasma substitutes. Ali- quots of 1.0 per cent solutions of each dextran in 0.05 M sodium acetate were treated with accurately measured volumes of methanol to give a series of mixtures containing 40, 45, 50, and 65 per cent methanol by volume. The mixtures were held in a 5’ bath for 1 hour, then centrifuged at 5”. The sediments were analyzed for dextran content (3), and the percentages of the polysaccharide that were precipitated within these same ranges of methanol concentration were calculated, with use of the similarly deter- mined content of each original dextran solution as 100 per cent.
The data (Table I) show that the Streptococcus 50 product was not ex- tensively thrown out of solution by 40 (or 45) per cent methanol, in contrast to the more typical native dextrans included for comparison. The pattern
of precipitation instead resembled that of the plasma substitutes, except that a definitely larger proportion of the streptococcal product than of the clinical dextrans failed to be precipitated by 50 per cent methanol. This behavior is unique for a native dextran. Among the dextrans isolated from cultures of more than 90 different bacteria by workers at the North- ern Utilization Research Branch of the Department of Agricult,ure, the products from only two strains (NRRL B-742 (9-11) and NRRL B-1443 (12)) failed to be precipitated almost completely by 42 per cent ethanol;
TABLE I Precipitability by Methanol of Streptococcus 60 Native De&ran, 18S,
Compared with That of Other Deztrans
Percentage of dextran precipitated within different ranges of methanol concentration
Type and source of dextran I -
040 .
Native, unfractionated Streptococcus DS, 50 (Preparation 1X3). L. mesenteroides, Bt . . “ “ NRRL B-512 substrainj A. capsulatum (3).
The bibliographic reference is given in parentheses. * An additional 18 per cent was unprecipitated by 65 per cent methanol. t 53 per cent ethanol had been used in isolating this dextran (4) to insure obtain-
ing the entire native product. $ Starting material for the preparation of clinical dextran was supplied by the
Commercial Solvents Corporation. $ Clinical dextrans were supplied (1951) by A. B. Pharmacia, the Dextran Cor-
poration, and the Commercial Solvents Corporation.
even these two dextrans were fully precipitated by 45 per cent ethanol and proved to be of high molecular weight.
Viscosity-Comparison of cleared solutions (0.50 per cent in 0.05 M sodium acetate) of Preparation 1% and of three other native dextrans at 30” in Cannon-Fenske No. 100 viscometers showed the inherent viscosity of the Streptococcus 50 product to be significantly lower than that of the others, which included an “autolyzed” L. mesenbroides B-512 dextran kindly supplied by Dr. Allene Jeanes. The values for vi (c = 0.50) were Preparation 18S, 0.28; L. mesenteroides B-512 Preparation C (13), 0.68; AC&&Z&T capsulutum dextran (3), 0.72; L. mesateroides B dextran (4),
1.33. The intrinsic viscosity [v] of the native streptococcal dextran was found to be 0.25, i.e. in the range prescribed for clinical type materials and well below the values found by Hellman (9) and by Jeanes et al. (14) for all but a few fractions among a large collection of native dextrans.
Serological Reactivity-In tests for precipitating capacity versus pneumo- coccus rabbit antisera known to be highly reactive with various native and clinical dextrans (2-5), Preparation 18’S gave visible react’ions with type 2 and type 20, but not with type 12 antiserum. The streptococcal product thus may be classed as a dextran of “serological type R” (4). As antici- pated from the original observations made on crude Streptococcus 50 culture fluids, visible precipitation of solutions of 185 required 2 to 4 times more type 2 or type 20 antibody than sufficed to precipitate solutions of most natural dextrans, such as those listed in Table I. Similarly diminished precipitating capacity is shown by partly hydrolyzed dextrans (5) and by certain native dextrans possessing low proportions of 1,6-linkag~.~ As will be clear from experiments that follow, the reduced serological capacity of Streptococcus 50 dextran is attributable to low molecular weight and not to any deficiency in 1,6-linkages. The product is free of levan, i.e. no precipitation occurred in tests with a potent levan-reactive antiserum (15), under conditions in which as little as 1 part of levan in 1000 parts of dextran would have been detected.
“Clinical Sized” Middle Fraction of Streptococcus 60 Dextran
Further information on the apparent capacity of Streptococcus 50 t,o produce dextran of clinical size was sought by examining that portion of the product which precipitated within methanol concentration limits of 43 and 50 per cent by volume. One sample of this “mid-fraction” was obtained, with two “end fractions,” from Preparation 18s; a second was isolated from additional cultures of Streptococcus 50.
Mid-Fraction Preparation 18SII-To 1600 ml. of a neutral 5.0 per cent solution of 18S, 1200 ml. of methanol were added. The mixture was kept at 25’ for 2 hours, then centrifuged. The sediment (43 per cent methanol- precipitable fraction, 18SI) was washed with methanol and dried in vacua over CaC12; yield, 13.6 gm. (moisture-free basis) or 17 percentof the original material. Treatment of the supernatant fluid with additional methanol to a concentration of 50 per cent by volume gave the mid-fraction, 18SI1, as a precipitate. This weighed 39.1 gm. (moisture-free basis) and repre- sented 49 per cent of the original native material. A final fraction, 18SII1, was precipitated between 50 and 65 per cent methanol; yield, 17.2 gm. or 21.5 per cent of the starting material.
Mid-Fraction Preparation d/t--3 liters of Streptococcus 50 culture grown
in a medium of 20 per cent sucrose, 0.75 per cent K2HPOd, and 0.22 per cent soluble corn steep-solidsa for 3 days at 37” were used. After adjust- ment to 25” and pH 7.0, methanol was added to 35 per cent concentration, and bacterial cells and insoluble gum were removed by centrifugation. All material precipitating at 55 per cent methanol concentration was then separated from the supernatant fluid, reprecipitated once from aqueous solution by methanol at 55 per cent concentration, washed with methanol, and dried; yield, 75 gm. A neutral 5 per cent solution of the dried product, fractionated between 43 and 50 per cent methanol, gave 41.5 gm. (moisture- free basis) of the mid-fraction.
General Properties of 18SII and ,%$--Some of the characteristics of the
TABLD II Properties of Mid-Fraction Preparations of Streptococcus 60 Deztran
Property* 1lSII 24
Yield, gm. per titer, of culture. . _ 19.1 13.8 Optical rotation, [a]&~, degreest +200.3 +194.5 Intrinsic viscosity, [v], dl. per gm.$. . 0.24 0.23 From periodate oxidation analysis (17)
The bibliographic reference is given in parentheses. * Moisture-free basis. t c = 1.0 (HZO), length = 2 dm.; thanks are due to Dr. V. du Vigneaud for use
of the polarimeter. $ Filtered solutions in 0.05 M sodium acetate were measured at 30” in Ostwald-
Cannon-Fenske No. 100 viscometers.
above 43 to 50 per cent mid-fraction preparations are summarized in Table II. The over-all yields, 19.1 and 13.8 gm. per liter of culture, represent 20.2 and 14.6 per cent of t,he amount theoretically obtainable (16) from the sucrose used. From their high positive optical rotations and behavior on periodate oxidation (17), it is clear that 18SII and 24 are dextrans with high proportions of 1,6-linkages. The modes of isolation and the intrinsic viscosity values suggest a molecular size generally similar to the clinical dextrans. Though not included in Table II, the results of serological tests also show that Preparations 18SII and 24 closely resemble the plasma sub- stitutes in having a more limited capacity to give visible precipitation with pneumococcus type 2 and type 20 rabbit antisera than ordinary native dextrans with correspondingly high proportions of 1, G-linkages. The cor-
3 Corn steep-liquor (about 26 per cent soluble solids) was kindly supplied by Mr. A. C. Hopkins, American Maize-Products Company.
rectness of these indications of molecular size was fully confirmed by de- terminations of molecular weight carried out by Dr. F. R. Senti and Dr. N. N. Hellman and described in this paper.
Lack of Reducing Cup&&-Molecules of acid-hydrolyzed dextrans possess an actively reducing glucose terminal unit, measurement of which permits calculation of the number average molecular weight, zn (18). Molecules of the streptococcal dextran evidently have, in place of a reducing terminal group, a non-reducing p-D-fructofuranosyl unit that can be split off by invertase action.
Mixtures containing 3.0 ml. of a 10.0 per cent aqueous solution of dex- tran (Preparations 18SI1, 24, or commercial plasma substitute) plus 1.0 ml. of purified yeast invertase in 0.1 M acetate buffer (pH 4.5) or 1.0 ml. of the buffer without enzyme, were placed in a bath at 46”. After 24 hours incubation, when the slow enzymatic action had reached completion, analyses were made for total reducing power, calculated as maltose (19,20), and for the content of ketose sugar which remained soluble on treatment of the mixtures with ethanol to 90 per cent concentration (21) ; the values obtained were used to calculate apparent reducing end group concentra- tions and approximate z,, values for the dextrans. The mixtures were also subjected to multiple ascending paper chromatography in 6:4:3 n-butanol-pyridine-water (22) to obtain information on the nature of any sugar present.
It is apparent (Table III) that the streptococcal dextrans in buffer showed little reducing power compared to the acid-hydrolyzed product of similar XL. Calculations made on the assumption that a reducing end group is present led to number average molecular weights (M, > 140,000) more than twice the weight average molecular weights determined by light scattering (M, = 54,300; SO,lOO), an impossible relationship on theoretical grounds (23). The molecules thus seem to lack a reducing end group, a condition that might have some advantage in a clinical dextran (24).
When incubated with yeast invertase, the streptococcal preparations showed a great increase in reducing power. Roughly half of the new re- ducing power was due to a ketose sugar, further identified on chromato- grams as fructose, which was present in 90 per cent ethanol extracts of the invertase-dextran mixtures and which evidently had been released from the dextran molecules.” The remainder of the reducing power could be attributed to newly acquired reducing end groups of the dextrans, since
4 An origin for the fructose in some accompanying substance rather than in the dextran itself seema most unlikely. The results of serological tests exclude the pos- sibility that traces of levan might be the source, while solubility considerations as well aa the absence of glucose on chromatograms which show the liberated fructose (Table III) exclude the possibility that traces of sucrose might be the source.
it permitted calculation of number average molecular weights (a, = 31,100; 39,200) now suitably related to the molecular weights determined by light scattering. The new values approach those of the hydrolyzed clin- ical dextran, which showed no change on incubation with invertase.
Molecules of the Streptococcus 50 dextrans thus appear to possess a non- reducing /3-n-fructofuranosyl terminal, the removal of which by yeast 8-nfructofuranosidase action leaves the polymer with a new, reducing
TABLE III
Action of Yeast Znvertase on Mid-Fraction Preparations of Streptococcus 60 Dextran
fraction 24 L. mesenteroides Acid-hydrolyzed clinica
dextrans
T
I
-
Invertase Buffer
Invertase Buffer
Invertase Buffer
_-
-
Reducing mwer, a! maltose
/ k
(A)* (BY
49.3 9.2
47.3 9.0
33.7 35.3
__
-
T
17.2t 2.3
22.3t 1.9
0.1 0.4
-
S‘ 0
/
_ .
-
Reducing Mol. wt., %, :nd group from reduciol f dextran end group
(A - B)* (*r-B)
32.1 31,100 6.9 (145,000)
25.3 39,200 7.1 (141,000)
33.6 29,300 34.9 28,700
Mol. wt., ia
by I& scattering
l The values are given in micromoles per gm. of dextran corrected for slight re- ducing power of invertase.
t Chromatograms showed a single sugar, migrating and reacting as fructose; glu- cose was not detected. No sugar was found on chromatograms of the other (con- trol) mixtures.
$ Data of Dr. F. R. Senti and Dr. N. N. Hellman (see the text). Q Lot 252X1, derived from NRRL B-512 substrain, was kindly furnished by Mr.
M. L. Bachmann, the Commercial Solvents Corporation.
terminal unit. These features suggest that the molecules actually may terminate with a sucrose group, as is the case with a number of fructans and oligosaccharides. The high proportion of fructose (0.27 and 0.37 per cent) released from Preparations 18SII and 24 further differentiates these products from previously described bacterial dextrans.6 Jeanes et al. (14) have shown that the natural dextrans in a large series contained no more than about 0.02 per cent fructose. Slreplococcus 50 (NRRL B-1351) dextran was the sole exception. It had a high fructose content
6 J. Corman, C. S. Stringer, and H. M. Tsuchiya (personal communication, Dr. H. M. Tsuchiya) have found, in independent studies, that the low molecular weight dextran component formed by the action of L. mesenferoides NRRL B-512F dextran- sucraae upon 30 per cent sucrose also contains fructose, at least part of which is re- leased on treatment with yeast invertase but not by honey invertase.
(0.26 per cent) by the anthrone method (14), as observed in the present work.
Mode of Formation of Streptococcus 60 Dextran
The manner in which the unusual Streptococcus 50 dextran is formed was identified by studying the isolated dextran-synthesizing enzyme system (cf. (16)) of the culture, obtained as follows. Strain 50 was cultivated for 20 hours at 37”, without agitation, in a medium containing 10 per cent
TABLE IV
Comparison of Dettran Synthesis from Sucrose by Streptococcus 60 and L. mesenteroides B Enzymes
Mixtures of 3.0 ml. of enayme plus 3.0 ml. of 20 per cent sucrose in 0.1 M acetate buffer (pH 5.0) incubated 24 hours at 25”. Values are for products formed and are corrected for contents of similarly incubated mixtures with heat-inactivated en- zyme.
I Dutran I
source of enzyme
Streptococcus DS, 50. .................. L. mesenteroidee, B (16). ................
Precipitated between limits of
19.6 0.6
~-. mg. per ml. m*. per ml.
20.5 32.8* 13.4 , 15.21
The bibliographic reference is given in parentheses. * Chromatograma showed abundant amounts of fructoee and of a second reducing
sugar (see footnote 6) migrating at a rate intermediate between maltose and iso- maltose.
t Chromatograms showed an abundant amount of fructose and a barely detectable trace of a elowly migrating reducing sugar.
sucrose, 2 per cent K,HPO4, and 0.43 per cent corn steep-solids. The cul- tures were centrifuged to remove bacterial cells, and each liter of fluid was treated with 375 gm. of ammonium sulfate. An extract of the sediment was made with distilled water (20 ml. per liter of culture), dialyzed in the cold against 0.1 M acetate buffer (pH 5.0), and finally clarified by centrifu- gation.
Table IV illustrates that synthesis of dextran from sucrose catalyzed by this streptococcal enzyme differs from that brought about by a typical L. mesenteroides dextransucrase (16). Only a small part of the dextran pro- duced by the former was precipitated from solution by 40 per cent methanol, as in the living cultures, whereas nearly all of the dextran synthesized by the L. mesenteroides enzyme was precipitated under these conditions.
Moreover, the dextran formed by the streptococcal enzyme was accom- panied by large amounts of two reducing sugars, identified from their reac- tions and mobilities on paper chromatograms as fructose and (probably) leucrose, which is 5-0-n-glucopyranosyl-n-fructopyranose (25);s in con- trast, the dextran (13.4 mg. per ml., or 83 mM) formed by the L. mesen- teroides enzyme was accompanied by a closely corresponding amount of fructose (15.2 mg. per ml., or 84 mM> as virtually the sole by-product.
The same streptococcal enzyme preparation was without demonstrable degradative effect upon dextran. Mixtures with several native L. mesen- teroides dextrans in 1 per cent final concentration, incubated and analyzed along with those of the experiment in Table IV, showed no loss of dextran precipitable by 40 per cent methanol, or gain of polysaccharide precipitable in the range of 40 to 65 per cent methanol; moreover, no increase in reduc- ing power (19) occurred, and no sugar appeared on paper chromatograms made of the mixtures.
It would seem most probable that the Streptococcus 50 dextran owes both its relatively low molecular weight and its B-n-fructofuranosyl end group to synthesis by an enzyme that utilizes sucrose as an acceptor sub- strate much more readily than the usual dextransucrase. The reaction might be represented as
n glucose < > fructose + glucose < > fructose +
(glucose <)” .glucose < > fructose + n fructose
where n glucosyl units (from several hundred rather than many thousand sucrose molecules) are transferred to chains built on the glucose moiety of each initiator sucrose molecule. That the streptococcal enzyme has unusu- ally high affinity for acceptor substrates is further suggested by the forma- tion of an oligosaccharide by-product, apparently by the transfer of a glu- cosyl unit from sucrose to fructose (6),6 under conditions in which ordinary dextransucrase forms little or none of this by-product.
Note on Mobculu~ Weight and Ultracentrifugal Sedimentation Patterns of Streptococcus 60 Dextran
BY FREDERIC R. SENTI AND NISON N. HELLMAN
(From the Northern Utilization Research Branch, Department of Agriculture, Peoria, Illinois)
Molecular weights of the whole soluble native dextran of Streptococcus 50, as well ss of several fractions prepared from it, were determined by a
6 A sample of the impure sugar, separated from a streptococcal enzyme-sucrose digest, was kindly examined by Dr. H. M. Tsuchiya who found (personal communi- cation) its mobility on paper identical with that of an authentic sample of leucrose cm.
light scattering procedure, with the use of a slightly modified Brice-Phoenix photometer as described elsewhere (26). Found, for the whole native preparation 18S, & = 58,260; for fraction 18SI (0 to 43 per cent methanol precipitate from 18S, 17 per cent yield), a, = 199,000; for fraction 18SII (43 to 50 per cent methanol precipitate from 18S, 49 per cent yield), mW = 54,300; for fraction 18SIII (50 to 65 per cent methanol precipitate from 18S, 21.5 per cent yield), a, = 11,200. Thus, the average molecular
TABLE V Characterization of Mid-Fraction Preparations and of Their Subfractions
FIG. 1. Sedimentation diagrams of Streptococcus 50 dextran preparations as com- pared to clinical dextran. Experimental conditions: dextran in 0.4 per cent aqueous solutions, centrifuged 170 minutes at 47,760 r.p.m. (165,000 X g). Curve A = whole native dextran 185, Curve B = mid-fraction 18511, Curve C = clinical dextran, Commercial Solvents Corporation lot No. 252X1.
weight of the whole soluble dextran 18s is in the range designated by mili- tary purchase specifications as acceptable for clinical dextran (27). How- ever, the molecular weights of fractions 18SI and 18SII1, and their yields from 18S, indicate that the unfractionated native streptococcal dextran possesses too wide a molecular weight distribution for clinical dextran. Military purchase specifications require that the molecular weights of the highest and lowest molecular weight subfractions, in amounts not exceed- ing 10 per cent, should not exceed 200,000 or be less than 25,000, respec- tively.
On the other hand, as shown in Table V, the middle fraction 18SII of the streptococcal dextran had nearly the desired molecular weight distribu- tion. Its highest molecular weight subfraction in part.icular was well within the upper limit of tolerance. The same may be said for mid-fraction PreparaCon 24, which had been isolated from a separate culture containing corn steep-liquor rather than yeast extract as the source of nitrogen. Pos- sibly because it had been precipitated initially by 55 per cent methanol instead of by 65 per cent ethanol, Preparation 24 had a slightly higher molecular weight than 18SII.
The changes in molecular distribution resulting from fractionation of 18s dextran, and the approximate suitability of the distribution of 18SII for clinical dextran, were further confirmed by ultracentrifugal examina- tion. Sedimentation diagrams were determined by using a Spinco model E ultracentrifuge as reported elsewhere (26). Fig. 1 compares the sedi- mentation diagrams for l&S, 18SI1, and a commercial clinical dextran after centrifugation of 0.4 per cent solutions for 170 minutes at 47,760 r.p.m. (165,000 X g). Fractionation of 18s evidently removed much slow sedi- menting (presumably low molecular weight) material, leaving fraction 18SII very similar to the commercial clinical dextran.
SUMMARY
1. A non-hemolytic streptococcus from the blood of a patient with sub- acute endocarditis has been found to convert sucrose to a dextran that closely resembles (except for its somewhat wider molecular weight distri- bution) the plasma substitutes made from hydrolysates of ordinary dex- trans.
2. By fractionation between 43 and 50 per cent methanol, a portion amounting to half of the natural streptococcal product is obtained which is truly a dextran of clinical size as judged by optical rotation, behavior on periodate oxidation, intrinsic viscosity, serological reactivity, sedimenta- tion pattern on ultracentrifugation, and molecular weight distribution by light scattering. In so far as the molecular weight of this material (54,300; 60,100) reflects the fractionation procedure, it should be possible to recover in equally good yield material of somewhat larger size, if desired, by ad- justing the fractionation.
3. Molecules of the streptococcal dextran, unlike those of acid-hydro- lyzed dextrans, lack an actively reducing glucose terminal. In its place, they have a non-reducing p-n-fructofuranosyl unit (presumably linked as in sucrose) which can be split off by yeast invertase. Because of t,he ex- tremely large size of ordinary dextran molecules, no information had hitherto been obtained on the character of t,he “reducing end” of a natu- rally formed dextran.
4. Studies made with the separated, cell-free, dextran-synthesizing en-
zyme system of the streptococcal strain show that the dextran is formed by a limited polymerization reaction rather than by the enzymic degradation of a dextran of high molecular iveight. The streptococcal dextran ap- parently owes both its relatively low molecular weight and its p-n-fructo- furanosyl end group to synthesis by an enzyme able to use sucrose as an acceptor substrate much more readily than the usual dextransucrase.
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