1466
Macromolecules 1980,13, 1466-1471
(5) Atkins, E. D. T.; Parker, K. D.; Preston, R. D. Proc. R. SOC. London, Sect. B 1969, 173, 209. Atkins, E. D. T.; Parker, K. D. J. Polym. Sci., Part C 1969, 28, 69. (6) Yamakawa, H. Macromolecules 1975,8, 339. (7) Einaga, Y.; Miyaki, Y.; Fujita, H. J. SOC.Rheol. Jpn. 1977,5, 188. (8) Einaga, Y.; Miyaki, Y.; Fujita, H. J. Polym. Sci., Polym. Phys. Ed. 1979, 17, 2103.
(9) Zimm, B. H. J. Chem. Phys. 1946, 14, 164. (10) Yamakawa, H.; Fujii, M. Macromolecules 1973,6, 407. (11) Bluhm, T. L.; Sarko, A. Can. J. Chem. 1977,55, 293. (12) Yamakawa, H.; Fujii, M. Macromolecules 1974, 7, 128. (13) Yamakawa, H.; Yoshizaki, T. Macromolecules 1980, 13, 633. (14) Utiyama, H.; Sakato, K.; Ikehara, K.; Setsuiye, T.; Kurata, M. Biopolymers 1974,12, 53. (15) Godfrey, J. E.; Eisenberg, H. Biophys. Chem. 1976, 5, 301.
Triple-Helical Structure of (1--+3)-/3-~-Glucan~ Y. Deslandes and R. H. Marchessault* Department of Chemistry, Universite de Montreal, Montreal, Quebec, Canada H3C 3Vl
A. Sarko Department of Chemistry, College of Environmental Science and Forestry, State University of New York,Syracuse, New York 13210. Received February 20, 1980 ABSTRACT: The molecular and crystal structure of the anhydrous form of curdlan, a (1+3)-j+D-glucan of bacterial origin, has been determined by combined X-ray diffraction analysis and stereochemical model refinement. The anhydrous polymorph crystallizes as a triple-stranded helix in a hexagonal unit cell with parameters a = b = 14.41 f 0.05 8, and c (fiber repeat) = 5.87 0.05 A. The space group is B3and there is one helix per unit cell. As in the triple-helical (1+3)-fl-D-xylan, the three strands of the glucan helix are parallel, right-handed,611 helices repeating in 3c, and they are in phase along the helix axis. The hydroxymethyl group of each glucose residue is in the tg position, and the crystal structure is extensively hydrogen-bonded. The reliability of the structure analysis is indicated by the residual index R = 0.233.
*
Introduction Polysaccharides are important constituents of plants and microorganisms,where they perform a variety of functions. Among the latter, the structural function of cellulose in the p l a n t cell wall and the food-reserve role of granular starch are well-known. In many cases, the function of a polysaccharide appears to be determined by its structure in t h e solid state, i.e., its molecular conformation and crystal structure. A variety of conformational and crystalline forms of polysaccharides have been observed, including the less common multiple-helical forms. For example, double-helix crystal structures exist in native starch’ and in carrageenan2p3 and a triple-helix structure appears in the (1-+3)-&D-Xylan of some green algaeq4 A triple-helical s t r u c t u r e has been proposed for (1+3)-p-~-glucan?~a polysaccharide which occurs in many fungi, bacteria, plants, and algae and is known variously as curdlan, pachyman, laminaran, laricinan, callose, and paramylon. Its functions a p p e a r to range from the structural in plants and fungi to food reserve in the granules of paramylon, where it is also more than 90% crystal line.'^^ This polysaccharide exhibits interesting and useful gel proper tie^,^ and of additional interest is that curdlan has been reported to possess antitumor activity.1° In common with many other polysaccharides, (1+3)-& D-glucan crystallizes in different hydrates, not all of which are t h o u g h t to be triple-helical or triplexes of t h e same type. For example, curdlang is poorly crystalline in its native state, but upon annealing it yields two polymorphs: t h e “dry” form obtained in vacuo and the “hydrate” form obtained at 75% or higher relative h ~ m i d i t y . Sixfold, ~ triple-helical crystalline forms have been proposed for ?Part 13 in the series “Packing Analysis of Carbohydrates and Polysaccharides”. * To whom correspondence should be addressed at Xerox Research Centre of Canada, 2480 Dunwin Drive, Mississauga, Ontario, Canada L5L 1J9. 0024-9297/80/2213-1466$01.00/0
Table I Comparison of Observed and Predicted d Spacings ( A ) for the “Dry” Unit Cell of Curdlan hkl
obsd
pred
hkl
obsd
pred
100 110 200 120 300 220 130 400 23 0 140 101 111
12.6 7.25 6.26 4.73 3.60 3.60 3.46 3.12 2.86 2.72 5.32 4.53
12.48 7.21 6.24 4.72 3.60 3.60 3.46 3.12 2.86 2.7 2 5.31 4.51
201 121 301 131 401 002 102 112 202 122 302
4.26 3.67 3.38 2.98 2.75 2.90 2.84
4.27 3.68 3.36 2.98 2.76 2.935 2.86 2.72 2.66 2.49 2.40
2’69 2.52 2.40
these two ~ o l y m o r p h s , 5although ~~ various other structures, ranging from a single helix” to a sevenfold triplex,12have been put forth. In this study, a detailed X-ray crystallograpic refinement of the “dry” form of curdlan has shown it to be a sixfold, triple-helical structure, as suggested earlier. Experimental Section Commercially available curdlan powder (Takeda Chemical Co., Japan) was the source of (1+3)-fl-~-glucan. Oriented fibers were prepared from a 10% solution in dimethyl sulfoxide by extruding with a syringe into a methanol bath at room temperature. Fibers were washed with water, allowed to dry at constant length, and annealed under tension in a sealed bomb at 145 “C in the presence of water and this was followed by evacuation for 12 h at room temperature. The crystallinity of the resulting “dry” polymorph, as shown in Figure 1,was improved dramatically. The density of the fibers was measured by flotation in a p-xylene-chloroform mixture. The unit cell parameters of the crystal structure were refined by least-squares procedures, using 23 measured reflections. Relative intensities of the reflections were obtained from radial tracings of the X-ray films, recorded with a Joyce-Loebl microdensitometer. The areas under the tracings were resolved into
0 1980 American Chemical Society
Vol. 13, No. 6, November-December 1980
(l-S)-&~-Glucan 1467
b
I
Figure 2. Projection of the triple helix in the xz plane. The crystallographic repeat of the helix is of the molecular repeat.
Hydrogen atoms are omitted.
t
Figure 1. X-ray fiber diiirxtiun pattern d the "dry" iorm of rurdlan, remrded u,ith ~1 flat-plate camera. The fiber axis is vert ical. individual intensities with a least-squarescurve resulutinn program,13 followed by currections for Lorentz and polarization factnrs, unequal film-to-sampledisrances of the diflrarted rays, and arcing of the reflectionsP The square roots of the zorrectrd, integrared intensities constituted t h e relative structure factor aniplitudes. T w o unobserved reflections were assigned relative intensities of one-half t h e minimum observable value and were treated identically. Results and Discussion Diffraction Measurements. The least-squares refinement of unit cell parameters resulted in a hexagonal unit cell with dimensions o = h = 1&11 f 0.05 A and c (fiber axis, = 5.87 z 0.05 A. The calculated density of 1.55 g,cms for this unit cell is in good agreement with the experimental density of 1.53 g cmJ, measured for the highly crystalline paramylon granules? assuming six glucose residues in the unit cell. A comparison of the observed and calculated d spacings is shitwn in Table I. On the basis of the systematic extinction of'OO1 reflections when I = 2n + I and the absence uf a twotuld symmetry axis and of a mirror plane in a glucopyranose ring, the H3 space group was assigned. Accurdingly, a triplehelix model, consisting of three sixfold helical strands, earh with an advance per nionomer of 2935 A, is propwed. The individual strands of the triplex are parallel and are in phase along the fiher axis; thus the repeat of the helix occurs after two glucose residues of one strand, or 5.87 A. A right-handed form of such a structure is shown in Figure 2 and is justified over the left-hand model in the suhsequent pages. S t r u c t u r e Analysis a n d Refinement. Model Selection a n d Packing Refinement. As indicated hy the diffraction data and the density, each unit cell contained six glucose residues. which was cunsistent with one triple
Figure 3. Projection of the triple helix in the xy plane, showing the intrahelix hydrogen-bond scheme. Hydrogen atoms are omitted. helii per cell. All other models, such as single and double helices, could be eliminated either on the basis of inconsistency with the X-ray data or poor Only the probability of left- and right-handed triple helices was therefore evaluated as suitable models, using a stereochemical packing refinement method.15J6 In this refinement, models of sixfold helical chains, with a repeat per turn of 17.61 A, as required by the X-ray data, were established in accordance with 4C1glucose ring geometry and accepted bond lengths, valence-bond angles, and ring conformation angles. The triplex structure was generated from such chains by rotations of + E O and -120' about the helix axis. The complete helix was then refined by minimizing the function
where r;,B;, and 4; are the bond lengths, bond angles, and
1468 Deslandes, Marchessault, and Sarko
model
Macromolecules
Table I1 Possible Models of (1- 3)-p-~-GlucanDetermined by Packing Refinement helix bridge O( 6) rotn, H bonds and their 0-0 length,d A rotn,'" deg angle, deg deg PE Right-Handed 7 5 gt 36.8
1
5.5
119.4
2
6.2
118.1
3 4 5
34.1 36.1 31.2
119.0 118.5 117.7
6 7
10.3 10.5
119.6 117.8
Left-Handed -57 gg 27.2 181 tg 28.7
8 9
8.0 38.2
118.6 119.4
5 3 gt -18 gg
40.4 20.9
10 11 12
37.2 37.9 38.1
118.5 118.9 117.8
1 5 2 tg -150 tg -107 gg
31.0 31.0 45.2
-93 gg
1 3 8 tg -10 -1 20
52.8 18.6 44.2 55
0(4),-0(6), O( 6),-0(6), 0(6),-0(5), 0(6),-0(6), 0(4),-0(6), none 0(6),-0(5), 0(4),-0(6), none 0(6),-0(5), 0(4),-0(6), 0(5),-0(6), 0(4),-0(6), O( 5),-O( 6), none 0(4),-0(6), none
2.64 2.69 2.95 2.78 2.81 2.64 2.39
2.69 2.80 2.69 2.74 2.93 2.72
nonbonded contactse none 1
none 1 1
none none none none 1 1 1
a Helix rotation is 0" when 0 ( 3 ) , is a t ( 0 , - y , 2);positive rotation is clockwise looking down the c axis. O( 6 ) is at 0" PE (packing energy) is the value of when the bond sequence O( 5)-C( 5)-C(6)-0(6) is cis and gt = 60", tg = 180", gg = 60". Subscripts define strands 1, 2, and 3. Strand 1is defined by the helix the nonbonded term of eq 1,in arbitrary units. position (cf. footnote a of this table). Strands 2 and 3 are, respectively, obtained by rotations about the helix axis of strand 1 of 1 2 0 and -120". e See Table 111.
conformation angles, respectively, for the 1, m, and n corresponding variables in the monomer residue, while r@, Ooi, $oi and SDri, SDO;, SD@iare their standard values and standard deviations, respectively. The first three terms of this function thus represent the bond-length, bond-angle, and conformation-angle strains occurring during the adjustment of these variables. The remaining term approximates the nonbonded repulsion, with d , the distance between the nonbonded atoms i and j , do, the corresponding equilibrium distance, wi, the weight assigned to the contact in the s u r n m a t i ~ n ,and ' ~ 1/W the weight of the nonbonded contact term in the entire function Y. The standard valence bonds and angles for the p-D-ghcopyranose ring were those of Arnott and Scott.ls All of the bond lengths, valence-bond angles, and conformation angles were refined in this manner. In addition, the length of the virtual bond (i.e., the length of the glucose residue measured between two successive glycosidic bridge oxygens) was varied in steps of 0.005-0.05 8, for each model, and the refinement was started with the hydroxymethyl group in gg, gt, and tg rotational positions.lg Hydrogen bonds were identified when appropriate oxygenoxygen contact distances were in the range of 2.5Ck3.10 8,. The results of the packing refinement are shown in Tables I1 and 111. The optimum length of the virtual bond was 4.765 8, and 12 models, five right-handed and seven left-handed, were found residing in local energy minima. A variety of helix positions and O(6) rotations were present. Three criteria have been found useful in evaluating the probability of polysaccharide packing models: (1) the packing energy (PE in Table 11), (2) the number and nature of short nonbonded contacts, and (3) the number and characteristics of the hydrogen bonds. The probability of any model with P E exceeding approximately 20 units is generally q~estionab1e.l~For models with an acceptable PE, the minimum of short contacts and the maximum of hydrogen bonds determine the most probable model. On this basis, two of the models shown in Table I1 were judged to be the most probable: the right-handed model 3, with O(6) near tg, and the left-handed model 9, with O(6) near gg.
Table I11 Short Nonbonded Contacts Present in the Models Described in Table I1 limit,b A model 2 4 5
contact
dist, A extreme normal
Right-Handed 1.75 O(4 h-H( 6A), 1.83 0(4),-H(6A), 2.20 0(6),-H(6B),
2.20 2.20 2.20
Left-Handed 0(4),-H(6B), 2.03 2.20 interchain 11 H(6A),-H(6B), 1.76 1.90 12 0(4)1-0(6)2 2.02 2.70 Reference 22. See footnote d in Table 11. 10
2.40 2.40 2.40 2.40 2.20 2.80
X-ray Refinement. Even though only two probable models resulted from packing refinement, all 12 models of Table I1 were refined against the X-ray intensities. This was done in the interest of comparing the results of stereochemical and X-ray refinements. The latter was carried out essentially in the same manner as the packing refinement, except that only the conformation angles were refined, keeping the bond lengths and most of the bond angles constant at their optimum values determined by packing refinement. The crystallographic reliability index
where Fo and F, are the observed and calculated structure factor amplitudes, respectively, was minimized in the process. No temperature factor was used in the refinement. The value of P E was calculated for each model at the end of the refinement. The results, shown in Table IV, are noteworthy in two respects. First, only one structure emerged as the most probable one: the right-handed model 3 with O(6) near tg. Both its R value and P E were considerably lower than those of other models. Second, the most probable X-ray model was nearly identical with the best right-handed packing model, 3 of Table 11, which also possessed the lowest value of PE. This indicates that the most probable
(1+3)-P-D-Glucan 1469
Vol. 13, No. 6, November-December 1980 Table IV Results of X-ray Refinement of t h e 1 2 Models Shown in Table I1 helix rotn,‘ model deg
bridge angle, deg
O(6k rotn, deg
Rd
PEC
0.301 0.304 0.278 0.333 0.371
75.5 73.6 21.9 78.9 71.5
Left-Handed -69 gg 0.413 119.5 119.1 0.385 -189 tg 120.3 40 gt 0.431 121.6 -8 0.449 119.5 1 5 0 tg 0.405 121.6 0.437 -148 tg -90 gg 0.380 120.3 a-c See corresponding footnotes in Table 11. temperature factor.
39.7 55.3 61.0 44.9 29.6 47.0 64.7 No
1 2 3 4 5
6 7 8 9 10 11 12
5.6 7.5 33.6 34.3 32.7
Righ t-Handed 115.6 43 gt 119.4 -75gg 115.6 1 4 9 tg 117.4 30 gt 120.2 -110
8.7 12.1 9.9 40.2 39.3 39.9 40.1
X-ray model resides in the conformational and packing energy minimum. Final Refinement. The most probable right-handed triplex was subjected to a final refinement, in which the function O=fR”+ (1-nY (3) was minimized. In this function Y is the stereochemical packing function of eq 1, R” is the weighted reliability index R” = [C(wllF0l- I~c112)/C~lFo121”2 (4) where w is the weight of the reflection and the fraction f is chosen to weight the R If term more heavily than the Y term. Optimally, f = 0.985, which weights the Y term to the extent of approximately 2% of the total. (In eq 3, R” is expressed in percent and not as a fraction, e.g., 23.3 rather than 0.233.) The refined variables included the length of the virtual bond, all conformation angles, and the bridge angle. All bond lengths and other bond angles were kept constant at their optimum stereochemical values. The minimization of the function O thus guarded against the development of unreasonable conformation angles and, to a smaller extent, against short contacts. The weighted residual R If was chosen in favor of the unweighted residual R because the observed structure factor amplitudes of five unobserved or less reliable reflections were assigned “half-weight” in this refinement (the five reflections are identified in Table VII). In addition, the components B,, By, and B, of an anisotropic temperature factor h2a *2B, k2b*’ By l2c*ZBz B = ~+ +(5) 4 4 4 were refined in the last cycle of the refinement sequence, partially to correct for unequal X-ray scaling factors. In ~
d
F i g u r e 4. Projection of the unit cell in the ab plane, showing all hydrogen bonds. Hydrogen atoms are omitted and strands 1, 2, and 3 are labeled.
this equation, h, k , and 1 are reflection indices, a*, b*, and c* are reciprocal unit cell parameters, and B, = By for a hexagonal unit cell. The above temperature factor should not be considered a true anisotropic temperature factor; rather, it is used to correct for unequal X-ray scaling factors and the effects of other, isotropically applied corrections. The results of this refinement are shown in Table V (model C), along with the best models obtained from previous stereochemical and X-ray refinements (models A and B). Selected conformation angles and hydrogen bonds of the final structure are shown in Table VI and a comparison of the calculated and observed structure factor amplitudes is shown in Table VII. The atomic coordinates are given in Table VI11 and the unit cell structure is shown in projection in Figure 4. As indicated by the data in Tables V-VII, the final structure is marked by good stereochemical features, as well as a good agreement with experimental X-ray intensities. The rotational position of the helix in the unit cell is nearly identical with that predicted by stereochemical refinement. The virtual bond has decreased slightly in length from 4.765 to 4.741 A compared with the best packing structure, and the bridge angle has decreased to 110.4’. The O(6) rotational position is only 22’ from tg. The bond lengths and angles were nearly identical with the corresponding standard values, but the glucose residue is slightly flattened, with some ring torsion angles deviating up to 8’ from the standard values (cf. Table VI). However, this is considered acceptable in view of the range of values observed in sugar crystal structures.’* Similarly, the smaller than usual bridge angle can also be considered to be within the normal range of values seen for polysaccharides. As an example of even smaller bridge angles, the double-helical a- and @-amylosesboth have a bridge angle of 105’.
Table V Comparison of Refinement Results model
Cd
helix rotn,u bridge angle, deg deg 34.1 33.6 32.8
O(6)rotn,b deg
R
1 3 8 tg 149 tg 158 tg
0.278 0.233
119.0 115.6 110.4
u-c See corresponding footnotes in Table 11. C, final, combined refinement.
R”
0.227
temp factor B = 0.0 B, = 0.6, B y = 0.6, B, = 4.7
PE
18.6 21.9 18
A, stereochemical refinement only; B, refinement against X-ray data only;
1470 Deslandes, Marchessault, and Sarko
Macromolecules
Table VI Selected Conformation Angles (Deg) and Hydrogen Bonds ( A ) of the Final Structure conformation anglea
exptl value
standard value b
0(5)-C(l)-C(2)-C(3) C(l)-C(2)-C(3)-C(4) C(2)-C(3)-C(4)-C(5) C(3)-C(4)-C(5)-0( 5) C(4)-C(5)-O( 5)-C( 1) C(5)-0(5)-C(l);C(2) @ : H(1)-C( 1)-O( 3) -C(3)’ li, : C(l)-0(3)‘-C(3)’-H(3)‘
53.4 -45.4 45.2 -52.5 64.6 -65.1 29.1 9.6
56.0 -52.2 53.0 -55.4 61.1 -62.2
hydrogen bondC
length
O( 2)1---0(2)2 (intrahelix) O( 4),---0(6)2 O( 6)l ---CY612
2.72 2.71 2.75 a The conformation angle A( l)-A(2)-A( 3)-A(4) viewed from A(2) to A(3) is positive when A(4) is clockwise Reference 14. See footnote d in relative to A(1). Table 11. Table VI1 Calculated and Observed Structure Factor Amplitudes
hkl
Fo
FCa
100 110 200 (120,210) 300 220 (130,310) 400 (230,320) (140,410) 101 111 201 (121,211) 301 221 (131,311) 401 102 (112,202) (122,212) 302 00 1
87 55 90 (78,80)112 38 28 (45,69)83 5 (44,69)82 (74,27) 79 112 210 178 (129,131)184 96 78 (24,93)96 50 72 (129,19) 129 (48,74) 88 100
Meridional Reflections 0
00 2
28
003
0
1026 67 80 93 58 25 50 46 27b*c 92b 112 253 185 164 106 21 110 84 56 103 111 109 not obsd weak not obsd
a For combined reflections, Fc= ( X F ~ ~ ) ‘ ’ *Reflec. tions assigned half-weight. Nonobserved reflections.
The triple helix is extensively hydrogen-bonded, as shown in Figures 3 and 4 and Table VI. The three strands of the triplex are linked together through triads of strong hydrogen bonds between the O(2) hydroxyls, and the helices are linked together through equally strong hydrogen bonds involving the O(4) and O(6) hydroxyls. In this fashion, all hydroxyl oxygens participate in at least one hydrogen bond. The ring oxygen ( O ( 5 ) )does not appear to be hydrogen-bonded, but it is within 3.18 8, from the O(4) of the next glucose residue in the chain. Disorder. One reflection of the first layer line (d spacing = 4.79 A) could not be indexed with the hexagonal unit cell; it required a two-helix, orthorhombic unit cell, obtained by enlarging the hexagonal cell to twice its volume. Because this diffraction spot was broader than the other reflections and it did not appear in the powder di-
Table VI11 Cartesian Coordinates of the Asymmetric Unit atom
X
O(3) (71) C(2) (73)
-2.016 0.322 -0.179 -1.620 -1.903 -1.272 -1.311 -0.083 -3.305 0.118 -2.570 -0.150 0.420 -2.226 -1.492 -1.759 -0.545 -1.145
C(4) C(5) C(6) O(2) O(4) O(5) O(6) H(1) H(2) H(3) H(4) H(5) H(6A) H(6B)
Y
-3.130 -3.531 -2.993 -3.422 -4.890 -5.297 -6.785 -1.566 -5.122 -4.947 -7.212 -3.046 -3.384 -2.848 -5.479 -4.800 -7.050 -7.279
z
0.0
2.912 1.578 1.345 1.650 2.973 3.219 1.566 1.703 2.976 3.744 3.715 0.809 1.982 0.884 3.759 3.886 2.307
agram of the more crystalline paramylon, it was ignored. However, its origin may reside in some other helix models shown in Table 11, which, although less probable, are still possible. This suggests that a small fraction of an alternate helix may be present in the crystal structure, locally destroying hexagonal symmetry and perhaps crystallizing in the orthorhombic unit cell. The alternate unit cell contributes to a paracrystalline phase, thus reducing the crystallinity of curdlan.
Conclusion As expected, the triple-helix structures of (1-3)-/3-~glucan and (1+3)-P-D-Xylan are similar. Both are righthanded and both are internally stabilized by hydrogen bonds between O(2) hydroxyls. The presence of the hydroxymethyl group in the glucan does not affect the conformation of the triple helix, as compared with the xylan, because it is situated on the periphery of the helix. However, such a location of the hydroxymethyl group in the glucan permits the formation of additional hydrogen bonds, not possible in the xylan. The dihedral angles 4 and J/ of (1+3)-P-~-glucan (29, loo) are similar to, but not identical with, those reported for laminarabiose (28, -379, the P-(1+3)-linked dimer of glucose.20 This suggests that the triple helix does not affect short-range c ~ n f o r m a t i o nand ~ ~ results simply from favorable intermolecular interactions. It is also interesting that in the triple helix, the O(6) are in the tg position, which is seldom observed in carbohydrate single-crystal ~tructures.~~ In the case of paramylon, where the degree of crystallinity is at least twice as great as that of annealed curdlan fibers,’ the triple-helix organization is most probably nascent in origin. This means that biosynthesis and crystallization are likely to take place simultaneously,21as it is difficult to imagine how high crystallinity could be achieved in a crystallization of fully formed chains. The low degree of crystallinity found in precipitates obtained from dilute solutions of curdlan, and in the fibers extruded from more concentrated solutions, supports this. The overall similarity of the X-ray patterns of the ‘‘dry‘’ and “hydrate” forms of curdlan5 and the reversibility of the transformation lead to the conclusion that the “hydrate” form is also triple-helical. However, the change in the c-axis dimension from 5.86 to 18.6 A in the “hydrate” can only be explained if there is a loss of P63 symmetry as a result of the hydration. The role of water in this transformation remains to be determined. Similarly, the nature of the transformation and features of a sevenfold
Macromolecules 1980, 13, 1471-1473
triple helix,12presumed to occur during gelation, are under investigation.
(11) Harada, T. ACS Symp. Ser. 1977, No. 45, 265-83. (12) Kasai, N.; Harada, T. “Abstracts of Papers”, 178th National Meeting of the American Chemical Society, Cellulose, Paper,
Acknowledgment. This work is based on the Ph.D. dissertation of Y. Deslandes presented to the Chemistry Department, Universit6 de Montreal. The financial support of the Ogilvie Flour Mills Co. to Y. Deslandes as well as that of the Ministere de 1’Education du Quebec and the Canadian National Research Council is gratefully acknowledged. This study was also supported by the National Science Foundation (Grant CHE7727749).
(13)
References and Notes
(15) (16)
(1) Wu, H. C.; Sarko, A. Carbohydr. Res. 1978, 61, 7-25, 27-40. (2) Anderson, N. S.; Campbell, J. W.; Harding, M. M.; Rees, D. A.; Samuel, J. W. B. J . Mol. Biol. 1969, 45, 85-99. (3) Rees, D. A.; Welsh, G. E. Angew. Chem., Int. Ed. Engl. 1977, 16, 214-24. (4) Atkins, E. D. T.; Parker, K. D. J. Polym. Sci., Part C 1969,28, 69-81. (5) Marchessault, R. H.; Deslandes, Y.; Ogawa, K.; Sundararajan, P. R. Can. J . Chem. 1977,55, 3OC-3. (6) Bluhm, T. L.; Sarko, A. Can. J . Chem. 1977,55, 293-300. (7) Marchessault, R. H.; Deslandes, Y. Carbohydr. Res. 1979, 75, 231-42. (8) Barras, D. R.; Stone, B. A. “The Biology of Euglena”; Academic Press: New York, 1969. (9) Harada, T. Process Biochem. 1974, 9, 21. (10) Sasaki, T.; Abiko, N.; Sugino, Y.; Nitta, K. Cancer Res. 1978, 38, 279-83.
1471
(14)
(17) (18) (19) (20) (21) (22)
(23) (24)
and Textile Division, Washington, D.C., 1979; American Chemical Society: Washington, D.C., 1979; Abstract No. 68. Atkins, E. D. T.; Fulton, W. S. Preprints of the IUPAC International Symposium on Macromolecules, Florence, 1980; Vol. 2, pp 292-5. Sarko, A. w (Fortran least-squares curve resolution program); College of Environmental Science and Forestry: SUNY, Syracuse, N.Y. Sarko, A. FIBRXRAT (Fortran X-ray intensity correction program); College of Environmental Science and Forestry: SUNY, Syracuse, N.Y. Zugenmaier, P.; Sarko, A. Biopolymers 1976, 15, 2121-39. Sarko, A.; Zugenmaier, P. ~ ~ (Fortran 7 9 virtual bond refinement program); College of Environmental Science and Forestry: SUNY, Syracuse, N.Y. Zugenmaier, P.; Sarko, A. Acta Crystallogr., Sect. B 1972,28, 3158-66. Arnott, S.; Scott, W. E. J . Chem. SOC.,Perkin Trans. 2 1972, 324-35. Sarko, A,; Marchessault, R. H. J.Polym. Sci., Part C 1969,28, 317. Takeda, H.; Kayia, T.; Yasuoka, N.; Kasai, N. Carbohydr. Res. 1978, 62, 27-37. Wunderlich, B. Macromol. Phys. 1976,2, 189-98. Sundararajan, P. R. Ph.D. Thesis, University of Madras, Madras, India, 1969. Walton, A. G.; Blackwell, J. Biopolymers 1973, 34. Perez, S.; Marchessault, R. H. Carbohydr. Res. 1978, 65, 114-20. Marchessault, R. H.; Perez, S. Biopolymers 1979,18,236%74.
Vacuum Ultraviolet Circular Dichroism of Dextran Arthur J. Stipanovic and E. S. Stevens* Department of Chemistry, State University of New York at Binghamton, Binghamton, N e w York 13901
K. Gekko Department of Food Science and Technology, Nagoya University, Nagoya, J a p a n . Received April 29, 1980
ABSTRACT: Vacuum ultraviolet circulcr dichroism (VUCD) spectra were recorded for a series of native dextrans [ (1+6)-a-~-glucans] ranging in M , from 410 to 303 000. Films exhibit a positive CD band near 167 nm with [e] = +1630 deg cm2 dmol-’. Evidence was obtained for a positive band of greater magnitude in solution at approximately the same wavelength. The variation in molar ellipticity with molecular weight is equal to or less than 10% over the M , range studied.
Vacuum ultraviolet circular dichroism (VUCD) spectroscopy has recently been used to characterize carbohydrates and po1ysaccharides.l4 This technique is potentially of special importance for those molecules with no electronic transitions above 190 nm, such as dextran, a slightly branched (1+6)-a-D-glucan. One of us911 has previously studied the intrinsic viscosity and other physicochemical properties of dextrans as a function of molecular weight. The purpose of this study was to measure the molecular weight dependence of dextran chiroptical properties.
Experimental Section Dextran samples ranging in molecular weight (A?,)from 410 to 44 500 were obtained by fractional precipitation of acidhydrolyzed native dextran (Meito Sangyo Co., Ltd.) estimated to contain 96% (1+!5)-a-1inkages.l0 Pharmacia dextrans T10 (A?n = 5200) and T500 (M, = 303 OOO) were also examined. All samples 0024-9297/80/2213-1471$01.00/0
were readily soluble in water a t 20-25 “C. The VUCD spectrometer and typical operating conditions are described e l ~ e w h e r e .Each ~ sample was examined in aqueous solution (both H20 and DzO) and as an amorphous film. Solution measurements were made in fused-silica cells of 0.100- and/or 0.054-mm path lengths, with concentrations ranging from 10-20 mg/mL. Moisture content correqtions were made for each solution based on elemental analysis data (Galbraith Laboratories). Films were prepared by allowing a 0.1-mL drop of the above solutions to air-dry on a 1.9-mm-diameter CaF’z disk at 80 “C. Films cast in this manner did not crystallize or adopt a preferred orientation. Molar ellipticities, [e],were calculated on the basis of a monosaccharide molecular weight of 162.
Results Figure 1shows a_ctualtracings for a solution (top) and film (bottom) of M , = 4500 dextran; those spectra are typical of all solution and film spectra obtained in this study. The 175-nm cutoff for aqueous solutions in 50-pm 0 1-980 American Chemical Society
