- Email: [email protected]
Vacuum ultraviolet circular dichroism of (1 → 6)-β-d -glucan
Vacuum ultraviolet circular dichroism of (1 • 6)- -D-glucan Arthur J. Stipanovic and E. S. Stevens Department ~f Chemistry, State University of New York, Binghamton, New York 13901, USA
(Received 20 September 1979; revised 16 November 1979) V.u.c.d. spectra recorded./br.Jreshly prepared aqueous solutions of( 1--*6)-[J-D-glucan (pustulan) contained a single positive hand near 177 nm. This band was similar in position and magnitude to the single positive baml observed in the spectrum oJ(1 --*6)-a-l)-glucan (dextran). Pustulan solutions (20 mg/ml) were observed to gel with time at 10 C. Concurrently, a negative band at 190 nm developed in the pustulan v.u.c.d, spectrum lbllowed by a blue sh(/~ o/both hands with continued aging. Crystalline films o]pustulan yield .~pectra which resembled the blue shilted spectra q/aged gels. The time dependent development q/the negative hand was attributed to pustulan attaining a helical conlbrmation in solution, and the blue sh(lt to aggregation ~71'helices. Na + and Ca 2 + were.]bund to accelerate gelation presumably hy decreasing the actirity O/the aqueous solvent.
Introduction The utility of vacuum ultraviolet circular dichroism (v.u.c.d.) spectroscopy in the study of polysaccharide conformation has been well established in recent years I 5. Although v.u.c.d, is quite sensitive to conformational changes arising from such factors as solvation, complexation, and molecular aggregation, it is not yet possible to assign unambiguously a probable conformation to a polysaccharide merely by comparing its v.u.c.d, spectrum to that of another, well characterized, molecule. An approach of this nature has been successfully applied to polypeptides, however, for which the ~thelical and fl-sheet conformations yield characteristic c.d. spectra 6. We have undertaken a v.u.c.d, investigation of several glucan homopolysaccharides in an attempt to correlate any unique features of their spectra to molecular conformation and configuration at the anomeric centre. Having already obtained spectra for dextran ~, a (1 --+6)~t-D-glucan, we directed our attention in the present study to pustulan, a linear (1 -*6)-fl-D-glucan. A comparison of the v.u.c.d, spectra of these two naturally occurring materials was expected to reveal the effect of C(1) configuration on the glucan chromophore(s), at least for (1 -*6)-linkages. Furthermore, it was expected that the conformational changes occurring during the sol-gel transition of pustulan could be monitored with v.u.c.d., as has been accomplished for agarose 4 and alginate 5 in the laboratory.
Experimental Unbleached, partially acetylated pustulan extracted from the lichen P. papullosa was obtained from Calbiochem, San Diego, California. The following technical data was provided by Calbiochem: (a) pustulan is soluble in hot H20; (b) [~]~' = - 4 0 ; (c) acid hydrolysis yields only glucose; (d) enzymatic hydrolysis yields only gentiobiose; 0141 8130/80/040209 04502.00 © 1980 IPC Business Press
(e) 2~,0 aqueous solutions gel with time and cooling; (f) repeated freezing and thawing of solutions results in precipitation of the polysaccharide. Deacetylation (saponification) of native pustulan was accomplished by dissolving the material in 1~o aqueous N a O H at 90'~C and allowing the solution to stand at 5060'C overnight. Following neutralization with HC1, the brown coloured solution was bleached with NaCIO 4 yielding a faintly yellow solution. The bleached pustulan was precipitated with 95~o ethanol and collected by vacuum filtration. After several washings with alcohol, the precipitate was redissolved in hot water, allowed to cool, and dialysed using an Amicon ultrafiltration membrane apparatus. Solutions were then freeze-dried. The UM 10 membrane used in the dialysis was effective in rejecting a large percentage of the pustulan in solution. Amicon data indicate that the UM 10 will typically reject 90~o of a Pharmacia Dextran T 10 solute of Mw ~ 104. The molecular weight of our pustulan sample was, therefore, assumed to be greater than 104. The v.u.c.d, spectrometer, described elsewhere 9, was operated at a 3.2 nm spectral width, 30 s time constant and 1.0 nm min- 1 scan speed. Spectra of pustulan solutions and gels were recorded in fused silica cells of 0.05 to 0.10 mm path length. Solution concentrations were typically 10 20 mg/ml H20. Gel spectra were recorded in v.u.c.d, solution cells cooled to 1(~15:C while spectra were being recorded. Film specimens were prepared by allowing a 0.25-0.50 ml drop of a 10-15 mg/ml solution ofpustulan to air dry on a CaF 2 disc (1.9 cm diameter) at 80°C. X-ray diffraction revealed that these films were partially crystalline but were not preferentially oriented. Molar ellipticities were calculated based on a monomer molecular weight of 162. Acetylated pustulan was prepared from the freeze-dried polysaccharide by the formamide method of Jeanes and Wilham s. Solubility characteristics in trifluoroethanol and chloroform indicated a high degree of acetyl substitution 1°. Gentiobiose, a (l~6)-/~-D-linked dimer of glucose, was obtained from Pfanstiehl Laboratories and was used without further purification.
Int. J. Biol. Macromol., 1980, Vol 2, August
209
V.u.c.d. of (l-*6)-fl-D-glucan: Arthur J. Stipanovic and E. S. Stevens 180 n.m. A similar positive band of greater ellipticity ([-0] = 2.3 x l03 deg d m o l - 1) was observed for gentiobiose, the (l ~6)-fl-linked glucose dimer which is chromophorically similar to pustulan. With aging and the onset ofgelation, a second band of negative ellipticity develops near 190 nm in the spectrum of pustulan solutions. Further aging results in an intensification of both bands, and a blue shift of the entire spectrum by almost l0 nm. It should be noted that in Figure 1 the spectrum taken at 32 days corresponds to a gel specimen while the spectrum at 38 days was recorded after the polysaccharide had begun to precipitate. As illustrated in Figures 2 and 3, the development of the negative v.u.c.d, band at 190 nm is accelerated by the
1.0
7~
0.5 A
~
0.0
' o -0.5
-I.0
I 180
I 190
200
I 210
X(nm)
V.u.c.d. spectra of a 20 mg/ml pustulan solution. A, 0 days; B, 7 days; C, 17 days; D, 32 days; E, 38 days Figure 1
2.0
_...'
1.0 A
o.o
Results
Gelation properties of pustulan With aging at 10°C for 5-6 days, 20 mg/ml aqueous PUstulan solutions were found to gain viscosity and begin gelation. More rigid gels, resistant to short term flow, were obtained after 10-14 days. A contraction of these gels and corresponding exclusion of solvent (syneresis) was observed after 17-21 days.of total aging. During the same time period, 10 mg/ml solutions increased in viscosity until 'islands' of gel were precipitated from the aqueous media. In both cases, gelation was thermally reversible with an observed melting temperature near 65°C. Once melted, gels did not reset unless the temperature was lowered to < 30°C. After longer periods of aging, ranging up to 40 days, gels of pustulan were found to lose viscosity suddenly, resulting in a suspension of precipitated particles of the polysaccharide. These particles, determined to -be crystalline by X-ray diffraction, could be redissolved in water at 80-90°C.~The resulting solution was still able to form a gel with appropriate aging, implying that the destruction of the gel network was not the result of microbiological degradation of the polysaccharide. In the presence of Na +, added as NaCI, the gelation rate of pustulan was found to accelerate. Solutions (20 mg/ml) containing equimolar quantities of Na ÷ and glucose monomer residues set to rigid gels after 2 days at 10°C. Lower concentrations of salt yielded proportionally lower extents of gelation enhancement. Ca 2+, at similar concentrations, added as CaCl 2, had a significantly greater s 'a gel promoter. Within five days, a gel was solvent excluded, and viscosity reduced as the haride precipitated.
'Pustulan v.u.c.d, spectra Viu.c.d. spectra recorded periodically over 38 days for a 20 mg/ml pustulan solution/gel are shown in Figure 1. Aging was performed at 10°C while the sample remained in an optical cell. Freshly prepared pustulan solutions (0 days, Figure 1) exhibit a single positive v.u.c.d, band near
210
Int. J. Biol. Macromol., 1980, Vol 2, August
'o
-I.0
- 2.0
I
I
I
I
190
200
210
220
X(nm)
Figure 2 . V.u.c.d. spectra of a 20 mg/ml pustulan solution with 0.5 C a 2 +/glucose monomer. A, 0 days; B, 2 days; C, 3 days; D, 8 days
2.0
'
1.0
A
B 'o
-IO
-2.0
I 180
1 190
l 200
I 210
I 27'0
k(nm)
Figure 3 V.u.c.d.spectra of a 20 mg/ml pustulan solution with 0.5 Na +/glucose monomer. A, 0 days; B, 2 days: C, 4 days; D, I l days; E, 18 days
V.u.c.d. of (1--*6)-fl-o-glucan: Arthur J. Stipanovic and E. S. Stevens d m o l - 1 and a weaker positive band was located near 170 nm ([0] =2.82 x 103 deg cm 2 dmol -~. 2.0
Discussion i
.~
1.0
E ~E t) g o.o t-i
'0
-LO
-2.0
1
I
I
I
I
180
190
200
2 I0
220
Xinml
Figure 4 V.u.c.d.spectra of a 20 mg/ml pustulan solution with 1.0 Na+/glucose monomer. A, 0 days: B, 2 days: C, 7 days; D, 30 days presence Of Na ÷ o r C a 2 +. These cations also enhance the molar ellipticity of both bands in the pustulan spectrum relative to the salt-flee solution/gel at similar periods of aging. A comparison of Figures 2 and 3 reveals that Ca 2 + is approximately twice as effective in promoting the 190 nm band as is Na +. It is noteworthy that aging of the cation-containing gels did not result in a blue shift of the v.u.c.d, band extrema, as was observed for the salt-free samples. With Ca 2 +-cOntaining gels it was found that the sample located between the windows of the v.u.c.d, cell was not typical of the bulk gel contained in the same cell. After aging periods of 4-7 days, the bulk gel decreased in viscosity and the polysaccharide began to precipitate while the material between the cell windows remained in the gel state. Suspensions of this precipitate, when placed in other v.u.c.d cells, gave rise to blue-shifted spectra similar to those recorded for the salt-flee solution/gel at 38 days. Whether the lack of a time-dependent blue shift in Na +-containing gels is the result of a similar phenomenon is .not certain since in that case the appearance of the gel between the cell Windows is quite similar to that in the rest of the cell. A comparison ofFiqures 3 and 4 illustrates that the enhancement effect of Na + is approximately proportional to concentration in the range of 0.5 1.0 mole cation/mole glucose. Films of pustulan cast from 10 mg/ml aqueous solution crystallized if dried slowly. X-ray diffraction revealed that these films were unoriented and partially crystalline. The X-ray diagram recorded for these samples is shown in Fiqure 5. An interplanar d-spacing of ~0.8 nm was calculated for the innermost reflection. V.u.c.d. spectra of these films are similar to those obtained for aged gels (Fitdure 1, 38 days) in that they contain a negative band near 180 nm ( [ 0 ] = - 2 . 7 × 102 deg cm 2 dmo1-1) and a positive band near 168 nm ( [ 0 ] = 3 . 0 × 102 deg cm 2 d m o l - 1). Pustulan triacetate in trifluoroethanol solution gave two bands of opposite sign in its v.u.c.d, spectrum. The n ~7~* band characteristic of acetylated polysaccharides was observed near 200 nm ([0] = - 4 . 0 2 × 103 deg cm 2
A primary objective of the present study was to c o m p a r e the v.u.c.d, spectra of pustulan to those of dextran and to identify any spectroscopic features which could be related to configuration at C(1). The similarity between the spectra of these two glucans suggests that pustulan in unaged solutions may exist, as does dextran, in the random-coil conformation. Under these circumstances, anomeric configuration apparently has little effect on the v.u.c.d, spectra of (1 ~6)-linked glucans. The positive band at 177 nm in pustulan and dextran spectra may tentatively be assigned to the acetal chromphore since their triacetates, not capable of hydroxyl absorption,, also exhibit this band. Furthermore, all glucose-containing polysaccharides studied thus far by v.u.c.d. 1'7 exhibit a positive dichroism band, in the 170180 nm range, regardless of linkage or Configuration at C(1), which also suggests that this band is not related to molecular conformation. Spectra recorded for (1 ~ 3)-~-Dglucan and (1 ~3)-a-D-glucan show this band as well ~~. A significant difference between the spectra of the (1 --*6) anomers arises when pustulan solutions are allowed to gel. With aging, a negative v.u.c.d, band centred near 190 nm begins to develop in the pustulan spectrum. With continued aging both bands intensify and blue shift almost 10 nm after 3(L40 days at 10~C. Assuming that gelation requires the formation of gel-junction zones in which the polysaccharide must possess a regular, helical conformation, we conclude that pustulan gelation and the development of a negative dichroism band are results of pustulan taking up such a conformation. The intensifi~ cation of t h i s band with gel aging may arise as more molecules, and larger segments of individual molecules, adopt the preferred helical conformation. Alternatively, the development and intensification of the 190 nm absorption may result from a blue shift of the Positive band at 180 nm which uncovers negative dichroism. Such a shift might arise if the 0(5) atom of the acetal chromophore became involved in a hydrogen-bond
Figure 5 X-Ray diagram of a crystalline pustulan film
Int. J. Biol. Macromol., 1980, Vol 2, August
21i
V.u.c.d. of(1 ~6)-[3-D-qlucan: Arthur J. Stipanovic and E. S. Stevens as the pustulan molecule adopts a helical conformation. This hypothesis is less probable, however, based on the observation that upon aging the negative band increases in ellipticity to a greater extent than can be accounted for by eliminating the positive contribution at 190 nm. Coincident with the development of the second band in the pustulan spectrum, is a blue shift of both bands with aging. After 30 days at 10 C, gel spectra were qualitatively similar to those recorded for crystalline films of pustulan. This result strongly suggests that intermolecular association of helical molecules, presumably through hydrogen bonding, is responsible for the observed shift to lower wavelengths. The collapse of pustulan gels with further aging ( ~ 38 days) must be attributed to an increase in aggregation or 'microcrystallization" beyond that point at which the network is made soluble by randomly orienting segments of the polysaccharide. V.u.c.d. spectra recorded for precipitated pustulan dispersed in an aqueous medium are similar to those of crystalline films, an observation which supports the mechanism proposed above. The presence of Na + and Ca z+ in concentrations ranging from 0.5 1.0 mole cation/mole glucose was found to increase the rate at which pustulan solutions would gel, w i t h C a 2 + being significantly more effective. Since pustulan at neutral pH exists as an uncharged molecule, the gelation enhancemen, t effected by cationic species is different in this case from that dominant in promoting gelation of polyanions, e.g. alginate 5. Further, an 'eggbox' mechanism of gelation in which Ca z + acts as an intermolecular crosslink is not likely since gelation will occur in the absence of added salt. In the case of pustulan, cations apparently enhance gelation by lowering the activity of the aqueous solvent, thereby increasing the tendency of the molecule to form helical conformations which associate into gel-junction zones. Sucrose, known to induce gelation by decreasing water activity 12.13, also increased the rate at which pustulan formed a gel. The
212
Int. J. Biol. Macromol., 1980, Vol 2, August
increased effectiveness of Ca 2 + is consistent with ionic mobility data revealing that Ca 2 + contains in its primary shell of hydration nearly twice the number of water molecules as does Na + .4 In summary, we conclude that the development of the negative c.d. band coincides with helix formation, and the blue shift reflects helix aggregation.
Acknowledgements This work was supported, in part, by NSF Grant PCM 79-04293. Acknowledgement also is made to the donors of the Petroleum Research Fund, administered by the American Chemical Society for partial support of this research.
References l 2 3 4 5 6
Lewis, D. G. and Johnson, Jr, W. C. Biopolymers 1978, 17, 1439 Balcerski, J.S.,Pysh, E.S.,Stevens, E.S.,Chen, G.C. andYang, J. T. J. Am. Chem. Soc. 1975, 97, 6274 Burlington, L. A., Pysh, E. S., Stevens, E. S., Chakrabarti, B. and Balazs, E. A. J. Am. Chem. Soc. 1977, 99, 1730 Liang, J. N., Stevens, E. S., Morris, E. R. and Rees, D. A. Biopolymers 1979, 18, 327 Liang, J. N., Stevens, E. S., Frangou, S. A., Morris, E. R. and Rees, D. A. Int. J. Biol. Macromol. 1980, 2, 204 Tinoco, Jr, 1. and Cantor, C. R. Methods Biochem. Anal. 1970, 18, 180
7 8 9 10 11 12 13 14
Stipanovic, A. J., Stevens, E. S. and Gekko, K. Macromolecules in press Jeanes, A. and Wilham, C. A. J. Am. Chem. Soc. 1952, 74, 5339 Pysh, E. S. and Stevens, E. S. Annu. Rev. Biophys. Bioen.q. 1976, 5, 63 Lindberg, B. and McPherson, J. Acta Chem. Scand. 1954, 8, 985 Stipanovic, A. J. and Stevens, E. S. unpublished work Dea, I. C. M., Morris, E. R., Rees, D. A., Welsh, E. J., Barnes, H. A. and Price, J. Carbohydr. Res. 1977, 57, 249 Rees, D. A. and Welsh, E. J. An qew. Chem. Int. Ed. En,ql. 1977, 16, 214 Bockris, J. O'M. and Reddy, A. K. N. "Modern Electrochemistry', Plenum Press, New York, 1970
(Received 20 September 1979; revised 16 November 1979) V.u.c.d. spectra recorded./br.Jreshly prepared aqueous solutions of( 1--*6)-[J-D-glucan (pustulan) contained a single positive hand near 177 nm. This band was similar in position and magnitude to the single positive baml observed in the spectrum oJ(1 --*6)-a-l)-glucan (dextran). Pustulan solutions (20 mg/ml) were observed to gel with time at 10 C. Concurrently, a negative band at 190 nm developed in the pustulan v.u.c.d, spectrum lbllowed by a blue sh(/~ o/both hands with continued aging. Crystalline films o]pustulan yield .~pectra which resembled the blue shilted spectra q/aged gels. The time dependent development q/the negative hand was attributed to pustulan attaining a helical conlbrmation in solution, and the blue sh(lt to aggregation ~71'helices. Na + and Ca 2 + were.]bund to accelerate gelation presumably hy decreasing the actirity O/the aqueous solvent.
Introduction The utility of vacuum ultraviolet circular dichroism (v.u.c.d.) spectroscopy in the study of polysaccharide conformation has been well established in recent years I 5. Although v.u.c.d, is quite sensitive to conformational changes arising from such factors as solvation, complexation, and molecular aggregation, it is not yet possible to assign unambiguously a probable conformation to a polysaccharide merely by comparing its v.u.c.d, spectrum to that of another, well characterized, molecule. An approach of this nature has been successfully applied to polypeptides, however, for which the ~thelical and fl-sheet conformations yield characteristic c.d. spectra 6. We have undertaken a v.u.c.d, investigation of several glucan homopolysaccharides in an attempt to correlate any unique features of their spectra to molecular conformation and configuration at the anomeric centre. Having already obtained spectra for dextran ~, a (1 --+6)~t-D-glucan, we directed our attention in the present study to pustulan, a linear (1 -*6)-fl-D-glucan. A comparison of the v.u.c.d, spectra of these two naturally occurring materials was expected to reveal the effect of C(1) configuration on the glucan chromophore(s), at least for (1 -*6)-linkages. Furthermore, it was expected that the conformational changes occurring during the sol-gel transition of pustulan could be monitored with v.u.c.d., as has been accomplished for agarose 4 and alginate 5 in the laboratory.
Experimental Unbleached, partially acetylated pustulan extracted from the lichen P. papullosa was obtained from Calbiochem, San Diego, California. The following technical data was provided by Calbiochem: (a) pustulan is soluble in hot H20; (b) [~]~' = - 4 0 ; (c) acid hydrolysis yields only glucose; (d) enzymatic hydrolysis yields only gentiobiose; 0141 8130/80/040209 04502.00 © 1980 IPC Business Press
(e) 2~,0 aqueous solutions gel with time and cooling; (f) repeated freezing and thawing of solutions results in precipitation of the polysaccharide. Deacetylation (saponification) of native pustulan was accomplished by dissolving the material in 1~o aqueous N a O H at 90'~C and allowing the solution to stand at 5060'C overnight. Following neutralization with HC1, the brown coloured solution was bleached with NaCIO 4 yielding a faintly yellow solution. The bleached pustulan was precipitated with 95~o ethanol and collected by vacuum filtration. After several washings with alcohol, the precipitate was redissolved in hot water, allowed to cool, and dialysed using an Amicon ultrafiltration membrane apparatus. Solutions were then freeze-dried. The UM 10 membrane used in the dialysis was effective in rejecting a large percentage of the pustulan in solution. Amicon data indicate that the UM 10 will typically reject 90~o of a Pharmacia Dextran T 10 solute of Mw ~ 104. The molecular weight of our pustulan sample was, therefore, assumed to be greater than 104. The v.u.c.d, spectrometer, described elsewhere 9, was operated at a 3.2 nm spectral width, 30 s time constant and 1.0 nm min- 1 scan speed. Spectra of pustulan solutions and gels were recorded in fused silica cells of 0.05 to 0.10 mm path length. Solution concentrations were typically 10 20 mg/ml H20. Gel spectra were recorded in v.u.c.d, solution cells cooled to 1(~15:C while spectra were being recorded. Film specimens were prepared by allowing a 0.25-0.50 ml drop of a 10-15 mg/ml solution ofpustulan to air dry on a CaF 2 disc (1.9 cm diameter) at 80°C. X-ray diffraction revealed that these films were partially crystalline but were not preferentially oriented. Molar ellipticities were calculated based on a monomer molecular weight of 162. Acetylated pustulan was prepared from the freeze-dried polysaccharide by the formamide method of Jeanes and Wilham s. Solubility characteristics in trifluoroethanol and chloroform indicated a high degree of acetyl substitution 1°. Gentiobiose, a (l~6)-/~-D-linked dimer of glucose, was obtained from Pfanstiehl Laboratories and was used without further purification.
Int. J. Biol. Macromol., 1980, Vol 2, August
209
V.u.c.d. of (l-*6)-fl-D-glucan: Arthur J. Stipanovic and E. S. Stevens 180 n.m. A similar positive band of greater ellipticity ([-0] = 2.3 x l03 deg d m o l - 1) was observed for gentiobiose, the (l ~6)-fl-linked glucose dimer which is chromophorically similar to pustulan. With aging and the onset ofgelation, a second band of negative ellipticity develops near 190 nm in the spectrum of pustulan solutions. Further aging results in an intensification of both bands, and a blue shift of the entire spectrum by almost l0 nm. It should be noted that in Figure 1 the spectrum taken at 32 days corresponds to a gel specimen while the spectrum at 38 days was recorded after the polysaccharide had begun to precipitate. As illustrated in Figures 2 and 3, the development of the negative v.u.c.d, band at 190 nm is accelerated by the
1.0
7~
0.5 A
~
0.0
' o -0.5
-I.0
I 180
I 190
200
I 210
X(nm)
V.u.c.d. spectra of a 20 mg/ml pustulan solution. A, 0 days; B, 7 days; C, 17 days; D, 32 days; E, 38 days Figure 1
2.0
_...'
1.0 A
o.o
Results
Gelation properties of pustulan With aging at 10°C for 5-6 days, 20 mg/ml aqueous PUstulan solutions were found to gain viscosity and begin gelation. More rigid gels, resistant to short term flow, were obtained after 10-14 days. A contraction of these gels and corresponding exclusion of solvent (syneresis) was observed after 17-21 days.of total aging. During the same time period, 10 mg/ml solutions increased in viscosity until 'islands' of gel were precipitated from the aqueous media. In both cases, gelation was thermally reversible with an observed melting temperature near 65°C. Once melted, gels did not reset unless the temperature was lowered to < 30°C. After longer periods of aging, ranging up to 40 days, gels of pustulan were found to lose viscosity suddenly, resulting in a suspension of precipitated particles of the polysaccharide. These particles, determined to -be crystalline by X-ray diffraction, could be redissolved in water at 80-90°C.~The resulting solution was still able to form a gel with appropriate aging, implying that the destruction of the gel network was not the result of microbiological degradation of the polysaccharide. In the presence of Na +, added as NaCI, the gelation rate of pustulan was found to accelerate. Solutions (20 mg/ml) containing equimolar quantities of Na ÷ and glucose monomer residues set to rigid gels after 2 days at 10°C. Lower concentrations of salt yielded proportionally lower extents of gelation enhancement. Ca 2+, at similar concentrations, added as CaCl 2, had a significantly greater s 'a gel promoter. Within five days, a gel was solvent excluded, and viscosity reduced as the haride precipitated.
'Pustulan v.u.c.d, spectra Viu.c.d. spectra recorded periodically over 38 days for a 20 mg/ml pustulan solution/gel are shown in Figure 1. Aging was performed at 10°C while the sample remained in an optical cell. Freshly prepared pustulan solutions (0 days, Figure 1) exhibit a single positive v.u.c.d, band near
210
Int. J. Biol. Macromol., 1980, Vol 2, August
'o
-I.0
- 2.0
I
I
I
I
190
200
210
220
X(nm)
Figure 2 . V.u.c.d. spectra of a 20 mg/ml pustulan solution with 0.5 C a 2 +/glucose monomer. A, 0 days; B, 2 days; C, 3 days; D, 8 days
2.0
'
1.0
A
B 'o
-IO
-2.0
I 180
1 190
l 200
I 210
I 27'0
k(nm)
Figure 3 V.u.c.d.spectra of a 20 mg/ml pustulan solution with 0.5 Na +/glucose monomer. A, 0 days; B, 2 days: C, 4 days; D, I l days; E, 18 days
V.u.c.d. of (1--*6)-fl-o-glucan: Arthur J. Stipanovic and E. S. Stevens d m o l - 1 and a weaker positive band was located near 170 nm ([0] =2.82 x 103 deg cm 2 dmol -~. 2.0
Discussion i
.~
1.0
E ~E t) g o.o t-i
'0
-LO
-2.0
1
I
I
I
I
180
190
200
2 I0
220
Xinml
Figure 4 V.u.c.d.spectra of a 20 mg/ml pustulan solution with 1.0 Na+/glucose monomer. A, 0 days: B, 2 days: C, 7 days; D, 30 days presence Of Na ÷ o r C a 2 +. These cations also enhance the molar ellipticity of both bands in the pustulan spectrum relative to the salt-flee solution/gel at similar periods of aging. A comparison of Figures 2 and 3 reveals that Ca 2 + is approximately twice as effective in promoting the 190 nm band as is Na +. It is noteworthy that aging of the cation-containing gels did not result in a blue shift of the v.u.c.d, band extrema, as was observed for the salt-free samples. With Ca 2 +-cOntaining gels it was found that the sample located between the windows of the v.u.c.d, cell was not typical of the bulk gel contained in the same cell. After aging periods of 4-7 days, the bulk gel decreased in viscosity and the polysaccharide began to precipitate while the material between the cell windows remained in the gel state. Suspensions of this precipitate, when placed in other v.u.c.d cells, gave rise to blue-shifted spectra similar to those recorded for the salt-flee solution/gel at 38 days. Whether the lack of a time-dependent blue shift in Na +-containing gels is the result of a similar phenomenon is .not certain since in that case the appearance of the gel between the cell Windows is quite similar to that in the rest of the cell. A comparison ofFiqures 3 and 4 illustrates that the enhancement effect of Na + is approximately proportional to concentration in the range of 0.5 1.0 mole cation/mole glucose. Films of pustulan cast from 10 mg/ml aqueous solution crystallized if dried slowly. X-ray diffraction revealed that these films were unoriented and partially crystalline. The X-ray diagram recorded for these samples is shown in Fiqure 5. An interplanar d-spacing of ~0.8 nm was calculated for the innermost reflection. V.u.c.d. spectra of these films are similar to those obtained for aged gels (Fitdure 1, 38 days) in that they contain a negative band near 180 nm ( [ 0 ] = - 2 . 7 × 102 deg cm 2 dmo1-1) and a positive band near 168 nm ( [ 0 ] = 3 . 0 × 102 deg cm 2 d m o l - 1). Pustulan triacetate in trifluoroethanol solution gave two bands of opposite sign in its v.u.c.d, spectrum. The n ~7~* band characteristic of acetylated polysaccharides was observed near 200 nm ([0] = - 4 . 0 2 × 103 deg cm 2
A primary objective of the present study was to c o m p a r e the v.u.c.d, spectra of pustulan to those of dextran and to identify any spectroscopic features which could be related to configuration at C(1). The similarity between the spectra of these two glucans suggests that pustulan in unaged solutions may exist, as does dextran, in the random-coil conformation. Under these circumstances, anomeric configuration apparently has little effect on the v.u.c.d, spectra of (1 ~6)-linked glucans. The positive band at 177 nm in pustulan and dextran spectra may tentatively be assigned to the acetal chromphore since their triacetates, not capable of hydroxyl absorption,, also exhibit this band. Furthermore, all glucose-containing polysaccharides studied thus far by v.u.c.d. 1'7 exhibit a positive dichroism band, in the 170180 nm range, regardless of linkage or Configuration at C(1), which also suggests that this band is not related to molecular conformation. Spectra recorded for (1 ~ 3)-~-Dglucan and (1 ~3)-a-D-glucan show this band as well ~~. A significant difference between the spectra of the (1 --*6) anomers arises when pustulan solutions are allowed to gel. With aging, a negative v.u.c.d, band centred near 190 nm begins to develop in the pustulan spectrum. With continued aging both bands intensify and blue shift almost 10 nm after 3(L40 days at 10~C. Assuming that gelation requires the formation of gel-junction zones in which the polysaccharide must possess a regular, helical conformation, we conclude that pustulan gelation and the development of a negative dichroism band are results of pustulan taking up such a conformation. The intensifi~ cation of t h i s band with gel aging may arise as more molecules, and larger segments of individual molecules, adopt the preferred helical conformation. Alternatively, the development and intensification of the 190 nm absorption may result from a blue shift of the Positive band at 180 nm which uncovers negative dichroism. Such a shift might arise if the 0(5) atom of the acetal chromophore became involved in a hydrogen-bond
Figure 5 X-Ray diagram of a crystalline pustulan film
Int. J. Biol. Macromol., 1980, Vol 2, August
21i
V.u.c.d. of(1 ~6)-[3-D-qlucan: Arthur J. Stipanovic and E. S. Stevens as the pustulan molecule adopts a helical conformation. This hypothesis is less probable, however, based on the observation that upon aging the negative band increases in ellipticity to a greater extent than can be accounted for by eliminating the positive contribution at 190 nm. Coincident with the development of the second band in the pustulan spectrum, is a blue shift of both bands with aging. After 30 days at 10 C, gel spectra were qualitatively similar to those recorded for crystalline films of pustulan. This result strongly suggests that intermolecular association of helical molecules, presumably through hydrogen bonding, is responsible for the observed shift to lower wavelengths. The collapse of pustulan gels with further aging ( ~ 38 days) must be attributed to an increase in aggregation or 'microcrystallization" beyond that point at which the network is made soluble by randomly orienting segments of the polysaccharide. V.u.c.d. spectra recorded for precipitated pustulan dispersed in an aqueous medium are similar to those of crystalline films, an observation which supports the mechanism proposed above. The presence of Na + and Ca z+ in concentrations ranging from 0.5 1.0 mole cation/mole glucose was found to increase the rate at which pustulan solutions would gel, w i t h C a 2 + being significantly more effective. Since pustulan at neutral pH exists as an uncharged molecule, the gelation enhancemen, t effected by cationic species is different in this case from that dominant in promoting gelation of polyanions, e.g. alginate 5. Further, an 'eggbox' mechanism of gelation in which Ca z + acts as an intermolecular crosslink is not likely since gelation will occur in the absence of added salt. In the case of pustulan, cations apparently enhance gelation by lowering the activity of the aqueous solvent, thereby increasing the tendency of the molecule to form helical conformations which associate into gel-junction zones. Sucrose, known to induce gelation by decreasing water activity 12.13, also increased the rate at which pustulan formed a gel. The
212
Int. J. Biol. Macromol., 1980, Vol 2, August
increased effectiveness of Ca 2 + is consistent with ionic mobility data revealing that Ca 2 + contains in its primary shell of hydration nearly twice the number of water molecules as does Na + .4 In summary, we conclude that the development of the negative c.d. band coincides with helix formation, and the blue shift reflects helix aggregation.
Acknowledgements This work was supported, in part, by NSF Grant PCM 79-04293. Acknowledgement also is made to the donors of the Petroleum Research Fund, administered by the American Chemical Society for partial support of this research.
References l 2 3 4 5 6
Lewis, D. G. and Johnson, Jr, W. C. Biopolymers 1978, 17, 1439 Balcerski, J.S.,Pysh, E.S.,Stevens, E.S.,Chen, G.C. andYang, J. T. J. Am. Chem. Soc. 1975, 97, 6274 Burlington, L. A., Pysh, E. S., Stevens, E. S., Chakrabarti, B. and Balazs, E. A. J. Am. Chem. Soc. 1977, 99, 1730 Liang, J. N., Stevens, E. S., Morris, E. R. and Rees, D. A. Biopolymers 1979, 18, 327 Liang, J. N., Stevens, E. S., Frangou, S. A., Morris, E. R. and Rees, D. A. Int. J. Biol. Macromol. 1980, 2, 204 Tinoco, Jr, 1. and Cantor, C. R. Methods Biochem. Anal. 1970, 18, 180
7 8 9 10 11 12 13 14
Stipanovic, A. J., Stevens, E. S. and Gekko, K. Macromolecules in press Jeanes, A. and Wilham, C. A. J. Am. Chem. Soc. 1952, 74, 5339 Pysh, E. S. and Stevens, E. S. Annu. Rev. Biophys. Bioen.q. 1976, 5, 63 Lindberg, B. and McPherson, J. Acta Chem. Scand. 1954, 8, 985 Stipanovic, A. J. and Stevens, E. S. unpublished work Dea, I. C. M., Morris, E. R., Rees, D. A., Welsh, E. J., Barnes, H. A. and Price, J. Carbohydr. Res. 1977, 57, 249 Rees, D. A. and Welsh, E. J. An qew. Chem. Int. Ed. En,ql. 1977, 16, 214 Bockris, J. O'M. and Reddy, A. K. N. "Modern Electrochemistry', Plenum Press, New York, 1970