Xufeng Zhang Lina Zhang Xiaojuan Xu Department of Chemistry, Wuhan University, Wuhan 430072, China
Morphologies and Conformation Transition of Lentinan in Aqueous NaOH Solution
Received 26 February 2004; accepted 17 May 2004 Published online 21 July 2004 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bip.20112
Abstract: Molecular morphologies and conformation transition of lentinan, a ␤-(133)-D-glucan from Lentinus edodes, were studied in aqueous NaOH solution by atomic force microscopy (AFM), viscometry, multiangle laser light scattering, and optical rotation measurements. The results revealed that lentinan exists as triple-helical chains and as single random-coil chains at NaOH concentration lower than 0.05M and higher than 0.08M, respectively. Moreover, the dramatic changes in weight-average molecular weight Mw, radius of gyration 具s2典1/2, intrinsic viscosity , as well as speciﬁc optical rotation at 589 nm [␣]589 occurred in a narrow range of NaOH concentration between 0.05 and 0.08M NaOH, indicating that the helix– coil conformation transition of lentinan was carried out more easily than that of native schizophyllan and scleroglucan, and was irreversible. For the ﬁrst time, we conﬁrmed that the denatured lentinan molecule, which was dissolved in 0.15M NaOH to be disrupted into single coil chains, could be renatured as triple helical chain by dialyzing against abundant water in the regenerated cellulose tube at ambient temperature (15°C). In view of the AFM image, lentinan in aqueous solution exhibited the linear, circular, and branched species of triple helix compared with native linear schizophyllan or scleroglucan. © 2004 Wiley Periodicals, Inc. Biopolymers 75: 187–195, 2004 Keywords: lentinan; helix– coil transition; triple-helical chain; light scattering; atomic force microscopy; denatured–renatured history
INTRODUCTION Lentinan is a ␤-(133)-D-glucan having two ␤-(136)glucopyranoside branches for every ﬁve ␤-(133)glucopyranoside linear linkages isolated from an edible mushroom, Lentinus edodes.1,2 The chemistry structure of lentinan is similar to that of schizophyllan3,4 and scleroglucan,5 which both exist as triplehelical chains in aqueous solution. Conformation
analysis from X-ray ﬁber diffraction6 has predicted ﬁve models including one single helix, two double helices, and two triple helices for the crystalline lentinan. Saito et al. have reported that the order– disorder conformation transition of lentinan in aqueous NaOH solution occurred between 0.13 and 0.19M by 13 C-NMR technology.7 Recently, we have conﬁrmed that lentinan dissolves in 0.5M NaCl aqueous solution
Correspondence to: Lina Zhang; email: [email protected]
wh.hb.cn Contract grant sponsor: National Natural Science Foundation of China (NNSFC) and UGC-AoE Plant and Fungal Biotechnology Project of Hong Kong Contract grant number: 20074025 (NNSFC) Biopolymers, Vol. 75, 187–195 (2004) © 2004 Wiley Periodicals, Inc.
Zhang, Zhang, and Xu
predominately as triple-helical chains and in DMSO as single ﬂexible chains.8 Moreover, the conformation transition from triple helix to coil of lentinan in water/ DMSO mixtures occurred in a narrow range between 80 and 85 wt % DMSO aqueous solution, and was irreversible.9 Both lentinan and schizophyllan have clinical application for treatment of certain cancer.10 –13 Therefore, a basic understanding of the molecular morphologies, conformation and conformation transition of ␤-(133)-D-glucans from fungal origin is essential for the successful application. It has recently been reported that one very stiff triple-helical molecule can be dissolved in the strong power solvent to change into three random-coil chains, and subsequently renatured as triple strands in linear and circular forms.14 –16 The conformation transition of triple-helix to random-coil chain occurs at 0.18 – 0.24M NaOH for schizophyllan17,18 and 0.1– 0.2M NaOH for scleroglucan19 at room temperature. The transition can also take place in water at elevated temperature above 135°C.20,21 Usually, the transition is as a result of breaking of inter- and intramolecular hydrogen bonds of triple-helix polysaccharides, leading to the strand separation and chain winding. Whereas the conformation properties and transition of schizophyllan or scleroglucan have been widely studied, it is surprising that the solution properties of lentinan has showed comparatively little interest in the literature. Different biological test systems have shown optimal stimulation either by the single-stranded or the triple-helical conformation.22 The immunostimulatory effect is related to the triple helix state of the ␤-(133)-D-glucan, so it is important to know how to prevent the transition from triple helix to single or to change the denatured ␤-(133)-D-glucan into the triple-helical molecule. In this article, we reported the molecular morphology of lentinan characterized by atomic force microscopy (AFM) and the conformation transition as a function of NaOH concentration. The detailed characterization of the conformation transition of lentinan was performed by multiangle laser scattering (MALLS), viscometry, and optical rotation.
MATERIALS AND METHODS Sample Preparation Lentinan was isolated from fruiting bodies of Lentinus edodes (Fangxian of Hubei in China) by extraction with 1.25M NaOH/0.05% NaBH4 two times, and then precipitated with 36% acetic acid to remove ␣-(133)-D-glucan,
according to a previously reported method.8 The supernatant was subjected to the Sevag method to remove proteins, and treated with 30% H2O2 to decolorize prior to adjusting the pH of the solution to 8 –9 with NH3–H2O aqueous solution. Aqueous solution of the lentinan was dialyzed against distilled water for 6 days, and then precipitated with acetone to get the crude lentinan. To obtain pure sample, the crude lentinan was dissolved in distilled water by stirring at room temperature, and then acetone was added dropwise into the solution to get the cloudy mixture of acetone and water (1:1 by volume). The puriﬁed lentinan was isolated from the resulting mixture by centrifuging at 6000 rpm at 15°C , and then was washed several times with acetone. The colorless powdery precipitate was vacuum-dried overnight at room temperature to obtain the lentinan sample. The renatured lentinan was prepared from denatured lentinan, which had been dissolved in 0.15M NaOH aqueous solution at the concentration of 1.2 mg/mL to change into single random coil, by dialyzing against abundant water in a regenerated cellulose tube for over 7 days at ambient temperature (15°C).
Laser Light Scattering (LLS) Intensities of light scattered from lentinan in aqueous NaOH solutions at 25°C were measured by MALLS equipped with a He–Ne laser (⫽632.8 nm) (DAWN威 DSP, Wyatt Technology Co. USA) in the angles of 35°, 43°, 52°, 60°, 69°, 80°, 90°, 100°, 111°, 121°, 132°, and 142°. The angular and concentration dependences of scattered intensities were analyzed by using Berry’s square root plot,23 that is (Kc/R)1/2 vs sin2(/2)⫹kc. Here K is the optical constant, c the polymer mass concentration, k the “stretch factor,” which scales the contributions from c to be roughly equal to the contributions from sin2(/2), and R the reduced scattering intensity at scattering angle . The instrument was calibrated by the usual method, with toluene at 25°C as the reference liquid. The Rayleigh ratio of pure ﬁltered toluene at 25°C at 632.8 nm was taken to be 1.406⫻10⫺5 cm⫺1. The normalization of the detectors and determination of the interdetector volume were performed with standard monodisperse pullulan (Mw/Mn ⫽1.1) with the molecular weight of 1.18⫻104 g/mol that did not show angular dependence on the light scattering signal.24 Optical clariﬁcation of aqueous NaOH solutions of lentinan was made by ﬁltration through a 0.45 m ﬁlter (NYL, 13mm Syringe ﬁlter, Whatman, Inc., USA). The speciﬁc refractive index increment (dn/dc) of lentinan in water at 25°C was measured with an optilab refractometer (DAWN威 DSP, Wyatt Technology Co., USA) at 633 nm to be 0.145cm3 g⫺1 at 632.8 nm. The dn/dc value was also used to the lentinan in aqueous NaOH solution. The value is in good agreement with that of schizophyllan in 0.01N NaOH.17 Astra software was utilized for data acquisition and analysis.
Viscometry Zero-shear-rate viscosities for lentinan at a NaOH concentration (wNaOH) lower than 0.08M were measured with a
Morphologies and Conformation Transition of Lentinan low-shear four-bulb capillary viscometer at 25°C.8 For lentinan at wNaOH higher than 0.08M, the Ubbelohde capillary viscometer was used. Kinetic energy correction was always negligible. Huggins and Kraemer plots were used to calculate intrinsic viscosity  by
sp/c ⫽ 关兴 ⫹ k⬘关兴2 c 共lnr)/c ⫽ 关兴 ⫺ k⬙关兴2 c where k⬘ and k⬙ are constants for a given polymer at a given temperature in a given solution, sp/c the reduced speciﬁc viscosity, (lnr)/c the inherent viscosity.
Optical Rotation Measurements The speciﬁc optical rotation at wavelength of 589nm [␣]589 was measured on a Perkin Elmer 341 polarimeter in a jacketed standard cell (10 cm/6.2 mL, Perkin Elmer) for about 0.1% lentinan in aqueous NaOH solution. The lentinan was dissolved in pure water, and then was added with the stronger aqueous NaOH solution to obtain the desired wNaOH for optical rotation measurement. All the solutions were maintained at 25°C.
Characterization of AFM Microscopy Lentinan was dissolved at approximately 1 mg/mL in aqueous solution by drastically stirring for 24 h, then diluted with distilled water to polymer concentration of 5 g/mL, and ﬁltered through a 0.45 m ﬁlter (NYL, 13 mm Syringe ﬁlter, Whatman, Inc., USA). A 10-L drop was deposited onto freshly cleaved mica and allowed to dry in air for 1.5 h at room temperature in a small covered Petri dish prior to imaging with magnetically AC (MAC) mode AFM. The specimen was examined using a Picoscan atomic force microscopy (Molecular Imaging, USA) in a MAC mode with commercial MAClever II tips (Molecular Imaging, USA), with a spring constant of 0.95 N/m. A piezoelectric scanner with a range up to 6 m was used for the image. The scanner was calibrated in the xy directions using a 1.0 m grafting and in the z direction using several conventional height standards. The measurement was performed in air at ambient pressure and humidity and the image was stored as 256⫻256 point arrays.
RESULTS AND DISCUSSION Light Scattering Analysis Scattered intensities at any angle of are transformed into the excess Rayleigh ratio R and analyzed by the equation26 共Kc/R 兲 1/␣ ⫽ 共1/M w1/ ␣ 兲兵1 ⫹ 共1/3␣兲具s2 典q2 ⫹ 共2/␣兲MwA2 c其
with K ⫽ (22n2/NA4)(dn/dc)2. q ⫽ 共4 n/ 兲sin(/2) where Mw is weight-average molecular weight; 具s2典1/2, z-average mean square root radius of gyration; NA, Avogadro’s number; , the wavelength of incident light in a vacuum, and n is the refractive index of solution. For ␣ ⫽ 1, Eq. (3) reduces to the “Zimm” formalism. It is worth noting that when the molecular mass of sample is greater than 1 ⫻ 106 the plot of (Kc/R) vs sin2(/2) for experimental data becomes practically nonlinear in a broad angular range yielding 具s2典1/2 unreliably.26 Berry23 has proposed the root plot with ␣ ⫽ 2, namely, the [(Kc/R)1/2] vs sin2(/2) plot, in the presence of nonlinear scattering angular variation. Figure 1, a and b, illustrates the Zimm plots using a Berry formalism with ␣ ⫽ 2 for lentinan in the 0.15M NaOH and 0.02M NaOH solution. The scattering envelopes in Figure 1 show a nonlinear angular dependence of the scattering that have noticeable downward curvatures. Similar curvature was also found at other aqueous NaOH solutions. For high molecular weight sample, the phenomenon is normal27 and a second-order polynomial ﬁtting in the angular extrapolation to zero angle furnishes reliable Mw and 具s2典1/2 results.
wNaOH Dependence of Mw and 具s2典1/2 Figure 2 shows the angular dependence of [(Kc/ R)1/2]c⫽0 for the lentinan in aqueous NaOH solution. From the intercepts and initial slopes of the indicated dotted lines the values of Mw and 具s2典1/2 were evaluated. The wNaOH dependences of the Mw and 具s2典1/2 for lentinan in aqueous NaOH solution are shown in Figure 3, a and b, respectively. The Mw of lentinan at wNaOH lower than 0.05M are approximately 3 times as large as that at wNaOH higher than 0.08M. Moreover, the Mw of lentinan at wNaOH lower than 0.05M are consistent with that in pure water, suggesting that the triple-helical conformation of lentinan maintains intact when wNaOH is lower than 0.05M. According to the report by Kashiwagi et al.,17 the addition of NaOH to an aqueous solution of schizophyllan enhances the solubility of the triple helix, but leads to gradual degradation of the helix when wNaOH exceeds 0.1M. In Figure 3, both the Mw and 具s2典1/2 decrease sharply at wNaOH higher than 0.05M, and level off at wNaOH higher than 0.08M. The abrupt change in the Mw and 具s2典1/2 reﬂected the dissociation of the triple helix into single coil chains, since the leveling-off
Zhang, Zhang, and Xu
FIGURE 1 Zimm plots for the lentinan in 0.15M NaOH aqueous solution (a) and in 0.02M NaOH aqueous solution (b) at 25°C.
Morphologies and Conformation Transition of Lentinan
FIGURE 2 Angular dependence of (Kc/R)c⫽0 for the lentinan in the aqueous NaOH solution of the indicated NaOH concentration (wNaOH) at 25°C. The ordinate values in the respective NaOH concentrations are shifted by A as indicated.
values of Mw and 具s2典1/2 at wNaOH higher than 0.08M are comparable to those of lentinan in pure DMSO, in which lentinan exists as random coil chain.8,9 To clarify whether the change of Mw and 具s2典1/2 in the wNaOH range between 0.05 and 0.08M is a result of the alkaline degradation taking place in the primary structure, the [␣]589 of lentinan in aqueous NaOH solution was measured. Figure 3c shows the results of [␣]589 for lentinan as a function of wNaOH. The value of [␣]589 for lentinan in pure water is in good agreement with that reported by Maeda et al.28 The changes of optical rotations of lentinan with an increase of wNaOH is considered to be a result of chain conformation transition rather than alkaline degradation of the primary structure, since some degradated derivatives of low molecular weight from lentinan have been reported to show the same [␣]D as lentinan.29 Thus we conclude that the [␣]589 decrease at the range of wNaOH between 0.05 and 0.08M reveals an order– disorder transition (i.e., triple helix to single coil transition).
wNaOH Dependence of  The wNaOH dependence of  is shown in Figure 4. The transition of triple helix to single random coil occurs at 0.05– 0.08M NaOH for lentinan at 25°C is indicated. The result further supports the conclusion from light scattering and optical rotation analysis. Recently, Yanaki et al.19 have reported the depen-
dence of the  on the wNaOH for a scleroglucan sample F-1 in aqueous NaOH solution at 25°C. Their experimental data are shown by a dashed line in Figure 4. The value of  for the scleroglucan sample F-1 is constant up to wNaOH of 0.05M, and then decreases sharply at wNaOH of 0.1M and level off at constant value at wNaOH higher than 0.2M, indicating the dissociation of a higher aggregates at wNaOH of 0.05M and the breaking of the trimer into single random-coil chains at wNaOH of 0.1M. Obviously, there are great differences in the viscosity behavior between lentinan and scleroglucan. A two-step dissociation process, with a dissociation of a higher aggregate consisting of triple-helix chains, was observed while the triple-helix lentinan was dissociated into single coil with one-step dissociation process. Moreover, The result from the viscosity measurement indicated that the  values for lentinan at wNaOH higher than 0.08M are close to that in DMSO at 25°C. However, the  values for scleroglucan at the range of wNaOH between 0.08 and 0.1M far exceed that (1.8⫻102 cm3 g⫺1) in DMSO. Obviously, the change of  for lentinan at wNaOH of 0.05M is attributed to the dissociation of the triple helix into single coil chain. Similar to lentinan, the  curve for schizophyllan also comprises only one step at wNaOH of 0.18M, which represents the transition of triple helix to single random coil chains.17,18 As mentioned above, the wNaOH (0.05– 0.08M) for the helix– coil transition of lentinan is lower than that for schizophyllan (0.18 – 0.24M)17,18 and scleroglucan
Zhang, Zhang, and Xu
tinan was obtained from an alkaline extract (1.25M NaOH), exposing the polysaccharide to the conditions disrupting the triple-helical structure (denaturation history), then in the condition favoring the triple formation by dialyzing the denatured lentinan against water (renaturation history). On account of their similarity in chemistry structure and X-ray ﬁber diffraction, the difference in transition range is presumably resulted from the denaturation–renaturation history of lentinan.
Renaturation of Lentinan
FIGURE 3 The wNaOH dependence of Mw (a), 具s2典1/2 (b), and [␣]589 (c) for lentinan in aqueous NaOH solution at 25°C. Solid circle represents the value for the renatured sample from denatured lentinan by dialyzing against water, and semisolid circle is the value measured by size exclusion chromatography combined with LLS (SEC-LLS) for the lentinan in DMSO at 25°C.
(0.1– 0.2M),19 suggesting a difference in the intermolecular hydrogen bonds, binding the triple chains together, of the lentinan from those of schizophyllan and scleroglucan. Note that the lentinan sample has a denaturation–renaturation history1,8—that is, the len-
To investigate the renaturation process, the values of lnr/c for the denatured lentinan, dissolved in 0.15M NaOH solution at the concentration of 1.2 mg/mL and then neutralized by 1.0M HCl to below wNaOH of 0.05M, were measured to be almost constant (data shown by open triangle in Figure 4). Moreover, the value of lnr/c for the neutralized lentinan solution did not increase and was constant when the solution was kept at 25°C for 4 days, indicating that the denaturation lentinan molecule could not be renatured to triple-helical chains by HCl neutralization, and the helix– coil transition of lentinan in the aqueous NaOH solution is irreversible. Interestingly, our experimental results revealed that the transition from single coil to triple helix of the denatured lentinan could occur by dialyzing against abundant water in regenerated cellulose tube for over 7 days. We measured the Mw, 具s2典1/2 and  of the renatured lentinan, and results are shown in Figures 3 and 4 (solid circle and solid triangle), indicating that the Mw, 具s2典1/2, and  come back to the magnitudes of the triple-helix lentinan. However, the linear ␤-(133)-D-glucan from the Poria cocos mycelia exists as aggregate30,31 in aqueous solution by dialyzing against water in the same condition, indicating that the restructuration of disrupted single ␤-(133)-D glucan is very complicated and interesting. The mechanism of renaturation is not well known and still under further investigation. We should note that the lentinan samples with triple-helix conformation in 0.5M NaCl8 or at wNaOH lower than 0.05M have been already exposed to the denaturation–renaturation history. This further conﬁrmed that the denatured lentinan has been renatured to triplehelical conformation by dialyzing against water.
Molecular Morphologies of Triple Helix Figure 5 shows an AFM image of the lentinan sample in pure water. Tracings across individual lentinan molecules illustrate the ability of AFM to measure the thickness of macromolecular chains normal to the
Morphologies and Conformation Transition of Lentinan
FIGURE 4 Dependence of  on wNaOH for lentinan in aqueous NaOH solution at 25°C. The dashed line indicates the data of scleroglucan F-1 by Yanaki et al.5 Solid triangle represents the value for the renatured lentinan by dialyzing against water and open triangle, value for the lentinan in 0.15M NaOH neutralized by 1.0M HCl to various wNaOH.
surface of the mica substrate. An example is shown in Figure 5. The measured mean thickness of the lentinan is 1.12⫾0.3 nm averaged over hundreds of molecules. The value is approximately 65% of the triple-strand thickness of a lentinan molecule with a center-to-center spacing of 1.73 nm expected from X-ray diffraction.6 The chain thickness of lentinan measured by AFM is consistent with that (about 1.0 nm) of the native triple-helix chain of scleroglucan and schizophyllan,32–35 so it is very interesting that both linear and circular species in lentinan consist of triple-stranded chains. This discrepancy in expected chain thickness is typical of many other systems investigated,33,34,36 and the origins are under investigation. Interestingly, a mixture of linear and circular structures plus several branched structures is observed in Figure 5. It has recently been discovered that the polysaccharides of triple-helical structure can be dissociated into random-coil chains and subsequently renatured as triple strands in linear, circular and branched forms by annealing at high temperature (about 100°C).14 –16,35,37 Moreover, Stokke et al.16 have reported an electron micrograph of a powdered sample of lentinan dissolved in water, showing circular and linear species. The circular and branched species have been also observed at the denatured–renatured schizophyllan14 and scleroglucan33 samples.
However, the images obtained from the native schizophyllan and scleroglucan have shown that only the linear structures are present in native material.32–34 The result from AFM conﬁrms that the lentinan is a renatured triple helix. Usually, the existence of the circular structures was quite unexpected given the stiff nature of the linear triple helix. Thermodynamic equilibrium arguments16 suggest that the energetic penalty, if any, associated with bending the triple helix into a circle is compensated by conformational degeneracy of the circles: Equienergetic triplestranded circular structures reconstituted from homogeneous single strands exist for many possible relative locations of the chain ends along the circumference. Only one linear triple helix, completely in register, realizes the energetic beneﬁt of full interchain valence saturation. On the basis of the Mw of lentinan in aqueous solution (3.11⫻106) and the weight-average contour length (1410 nm, 100 molecules) measured from the AFM images similar to Figure 5, the ML (ML ⫽ Mw/L) was found to be 2200 ⫾ 200 nm⫺1. The ML value is consistent with the proposed triple-helical structure,6,8,38 – 41 indicating that the various species of lentinan in the AFM image consist of triple-helix chain. Stokke et al.14 have reported a micrograph of a schizophyllan circle, providing direct evidence for circular triple-stranded schizophyllan. It is also worth
Zhang, Zhang, and Xu
FIGURE 5 MAC mode AFM topographic image of the lentinan deposited on mica as a 5 g/mL solution in pure water (top). Scale bar ⫽ 600 nm. The z scale is as shown. The arrow A indicates the linear triple-helical chains. The arrow B indicates the circular triple-helical chain. The arrow C indicates the branched triple-helical chains. The sample was air-dried onto mica for about 1.5 h and imaged. Trace across through one cyclic lentinan chain shown above as horizontal white bar (bottom).
noting that the Mw of the renatured lentinan at wNaOH lower than 0.05M is approximately 3 times as large as that at wNaOH higher than 0.08M or in DMSO, providing more strong evidence that the linear or circular species in the renatured lentinan is triple stranded. The circular species are reminiscent of circular DNA42 and probably some biological signiﬁcance for lentinan is correlative with the circular triple-helical structure.
CONCLUSIONS The results described in this paper, including the changes of Mw, 具s2典1/2, , and [␣]589 of the lentinan in aqueous NaOH solution associated with the wNaOH,
indicated the induced helix– coil transition. On account of a denatured–renatured history, lentinan exists as linear, circular, and branched triple-helical chains at wNaOH lower than 0.05M, and as single random-coil chains at wNaOH higher than 0.08M. The helix– coil transition of the lentinan occurred sharply in the range of wNaOH between 0.05 and 0.08M, and is irreversible. However, The denatured lentinan molecule, which was dissolved in 0.15M NaOH aqueous solution to change into single-coil chain, could be renatured to the triple-helical conformation by dialyzing against abundant water in regenerated cellulose tube at ambient temperature (15°C) for over 7 days. This work was supported by the National Natural Science Foundation of China (20074025) and UGC-AoE Plant and
Morphologies and Conformation Transition of Lentinan Fungal Biotechnology Project of Hong Kong. We thank Professor D. Pan and Mr. Z. Lu for the favor with AFM measurement.
REFERENCES 1. Chihara, G.; Maeda, Y.; Hamuro, J.; Sasaki, T.; Fukuoka, F. Nature 1969, 222, 687– 688. 2. Sasaki, T.; Takasuka, N. Carbohydr Res 1976, 47, 99 –104. 3. Kikumoto, T.; Miyajima, T.; Kimura, K.; Okubo, S.; Komatsu, N. J Agric Chem Chem Soc Jpn 1971, 45, 162. 4. Tabata, K.; Ito, W.; Kojima, T.; Kawabata, S.; Misaki, A. Carbohydr Res 1981, 89, 121. 5. Yanaki, T.; Kojima, T.; Norisuye, T. Polym J 1981, 13(12), 1135–1143. 6. Bluhm, T. L.; Sarko, A. Can J Chem 1977, 55, 293– 299. 7. Saito, H.; Ohki, T.; Sasaki, T. Carbohydr Res 1979, 74, 227–240. 8. Zhang, L.; Zhang, X.; Zhou, Q.; Zhang, P.; Li, X. Polym J 2001, 33(4), 317–322. 9. Zhang, L.; Li, X.; Zhou, Q.; Zhang, X.; Chen, R. Polym J 2002, 34(6), 443– 449. 10. Taguchi, T.; Furue, H.; Kimura, T.; Kondo, T.; Hattori, T. Jpn J Cancer Chemother 1985, 12, 366. 11. Ito, H.; Yagita, A.; Watanabe, Y.; Kitajima, M.; Sohma, S.; Akima, K. Shokaki-to-Menaki 1985, 14, 263. 12. Jong, S.-C.; Donovick, R. Adv Appl Microbiol 1989, 34, 183–262. 13. Pretus, H. A.; Ensley, H. E.; McNamee, R. B.; Jones, E. L.; Browder, I. W.; Williams, D. L. J Pharm Exp Therap 1991, 257, 500 –510. 14. Stokke, B. T.; Elgsaeter, A.; Brant, D. A.; Kitamura, S. Macromolecules 1991, 24, 6349 – 6351. 15. Stokke, B. T.; Elgsaeter, A.; Brant, D. A.; Kitamura, S. In Physical Chemistry of Colloids and Interfaces in Oil Production; Toulhoat, H., Lecourtier, J., Eds.; Edition Technip.: Paris, 1992; pp 217–223. 16. Stokke, B. T.; Elgsaeter, A.; Brant, D. A.; Kuge, T.; Kitamura, S. Biopolymers 1993, 33, 193–198. 17. Kashiwagi, Y.; Norisuye, T.; Fujita, H. Macromolecules 1981, 14, 1220 –1225. 18. Bo, S.; Milas, M.; Rinaudo, M. Int J Biol Macromol 1987, 9, 153–157.
19. Yanaki, T.; Norisuye, T. Polym J 1983, 15, 389 –396. 20. Yanaki, T.; Tabata, K.; Kojima, T. Carbohydr Polym 1985, 5, 275–283. 21. Kitamura, S.; Kuge, T. Biopolymers 1989, 28, 639 – 654. 22. Bohn, J. A.; BeMiller, J. N. Carbohydr Polym 1995, 28, 3–14. 23. Berry, G. C. J Chem Phys 1966, 44, 4550. 24. Wyatt, P. J. Anal Chim Acta 1993, 1, 2721. 25. Einaga, Y.; Miyaki, Y.; Fujita, H. J Polym Sci, Polym Phys Ed 1979, 17, 2103. 26. Nakata, M. Polymer 1997, 38, 9. 27. Tsuboi, A.; Norisuye, T.; Teramoto, A. Macromolecules 1996, 29, 3597–3602. 28. Maeda, Y. Y.; Watanabe, S. T.; Chihara, C.; Rokutanda, M. Cancer Res 1988, 48, 671– 675. 29. Sasaki, T.; Takasuka, N.; Chihara, G.; Maeda, Y. Y. Gann 1976, 67, 191–195. 30. Zhang L.; Ding Q.; Zhang P.; Zhu, R.; Zhou, Y. Carbohydr Res 1997, 303, 193–197. 31. Ding Q.; Zhang, L.; Wu, C. J Polym Sci Part B: Polym Phys Ed 1999, 37, 3201–3207. 32. McIntire, T. M.; Brant, D. A. J Am Chem Soc 1998, 120, 6909 – 6919. 33. Mcintire, T. M.; Penner, R. M.; Brant, D. A. Macromolecules 1995, 28, 6375– 6377. 34. McIntire, T. M.; Brant, D. A. Biopolymers 1997, 42, 133–146. 35. Vuppu, A. K.; Garcia, A. A.; Vernia, C. Biopolymers 1997, 42, 89 –100. 36. Mu¨ller, D. J.; Engel, A. Biophys J 1997, 73, 1633– 1644. 37. Falch, B. H.; Elgsaeter, A.; Stokke, B. T. Biopolymers 1999, 50, 496 –512. 38. Stokke, B. T.; Brant, D. A. Biopolymers 1990, 30, 1161–1181. 39. Yanaki, T.; Norisuye, T.; Fujita, H. Macromolecules 1980, 13, 1462–1466. 40. Norisuye, T.; Yanaki, T.; Fujita, H. J Polym Sci, Polym Phys Ed 1980, 18, 547–558. 41. Bluhm, T. L.; Deslandes, Y.; Marchessault, R. H.; Perez, S.; Rinaudo, M. Carbohydr Res 1982, 100, 117. 42. Semlyen, J. A. Cyclic Polymer; Elsevier Applied Science: London, 1986
Reviewing Editor: Dr. C. Allen Bush