May/June 2002
Published by the American Chemical Society
Volume 3, Number 3
© Copyright 2002 by the American Chemical Society
Communications Thermally Reversible Hydration of β-Chitin Yukie Saito,* Hiroko Kumagai, Masahisa Wada, and Shigenori Kuga Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan Received November 20, 2001
Thermally induced transition between anhydrous and hydrated forms of highly crystalline β-chitin was studied by differential thermal calorimetry (DSC) and X-ray diffraction. DSC of wet β-chitin in a sealed pan gave two well-defined endothermic peaks at 85.2 and 104.7 °C on heating and one broad exothermic peak at between 60 and 0 °C on cooling. These peaks were highly reproducible and became more distinct after repeated heating-cooling cycles. The X-ray diffraction pattern of wet β-chitin at elevated temperature showed corresponding changes in d-spacing between the sheets formed by stacking of chitin molecules. These phenomena clearly show that water is reversibly incorporated into the β-chitin crystal and that the temperature change induces transitions between anhydrous, monohydrate, and dihydrate forms. The DSC behavior in heating-cooling cycles, including reversion between the two endothermic peaks, indicated that the transition between monohydrate and dihydrate was a fast and narrow-temperature process, whereas the one between the anhydrous and the monohydrate form was a slow and wide-temperature process. 1. Introduction Chitin is one of the most abundant structural biopolymers on the Earth, occurring as the structural component of fungi, insects, and crustaceans. It has several crystal forms, major of which are R- and β-chitin.1 β-Chitin, the rarer allomorph, occurs in squid pen, certain diatoms, and vestimentiferans (a class of deep-sea animal). β-Chitin has a one-chain unit cell with P21 symmetry with a parallel-chain structure,2 in contrast to the two-chain, antiparallel structure of R-chitin.3 A remarkable feature of β-chitin is that it incorporates small molecules into the crystal lattice to form various crystalline complexes (crystallosolvates).4,5 The most important and readily formed complexes are the hydrates. The early X-ray diffraction studies identified anhydrous, monohydrated, and dihydrated forms.2 The transitions between these forms take * To whom correspondence may be addressed: Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Phone: +81-3-5841-7551. Fax: +81-3-5684-0299. E-mail: aysaito@ mail.ecc.u-tokyo.ac.jp.
place rather easily through drying and wetting. Basic features of the transition and the structure of hydrates have been reported6 but with some confusion in interpretation of crystal forms and formation of hydrates. Later studies established the reversibility of hydration,2,7 but the nature of transitions has not been fully understood, perhaps because of experimental difficulties. We studied here, using highly crystalline β-chitin from a vestimentiferan, Lamellibrachia sp., the interconversion between the anhydrous and hydrated forms by differential thermal analysis (DSC) and X-ray diffraction. 2. Experimental Section 2.1. Chitin Sample. The tubes of Lamellibrachia sp., collected at depth of 1160 m offshore Hatsushima in Sagami Bay, Japan, were used. The tube was rinsed and deproteinized by 5% KOH and buffered 3.4% NaClO28 and freeze-dried. This sample is known to consist of highly crystalline β-chitin9 composed of microfibrils approximately 30 nm wide.
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Figure 1. DSC thermogram of wet β-chitin. The lower set shows heating curves to 150 °C, and the upper set shows cooling curves to -20 °C. All were at 20 °C/min heating/cooling. The cycle was repeated 11 times.
2.2. Differential Thermal Analysis (DSC). About 10 mg of the freeze-dried sample was thoroughly dehydrated in a vacuum at 105 °C overnight, weighed accurately, and rewetted with about 14 mg of water (weighed accurately) in a stainless steel sealing pan (60 µL, Perkin-Elmer; compressive strength of 24 atm, corresponding to water vapor pressure at 220 °C). DSC measurements were performed with a Pyris1 calorimeter (Perkin-Elmer) at a heating/cooling rate of 20 °C/min. 2.3. X-ray Diffraction. The dry β-chitin specimen was wetted with water, and excess water was removed by blotting. The wet specimen was packed in a glass tube of 0.5 mm i.d., which was flame-sealed immediately. The sealed tube was mounted on a heating holder equipped with a thermocouple. The diffraction pattern was recorded by a goniometer equipped with a one-dimensional positionsensitive proportional counter (PSPC, Rigaku RINT 2200). Ni-filtered Cu KR radiation (λ ) 0.15418 nm) generated at 36 kV, and 50 mA was irradiated through a collimator with a pinhole of 1.0 mm diameter. The diffraction pattern was recorded at 25, 40, 60, 80, 100, and 120 °C, with 3 min accumulation. Heating rate was 5 °C/min. 3. Results and Discussion 3.1. DSC. Figure 1 shows the thermograms of wet β-chitin (9.8 mg of dry chitin + 14.1 mg of water) by repeated heating/cooling cycles between -20 °C and 150 °C. The first heating from room temperature showed two partly overlapped endothermic peaks at 86.0 and 104.9 °C (temperatures for peak maxima, same in the following). Subsequent cooling gave one broad exothermic peak at between 60 and 0 °C. After keeping at -20 °C for 5 min, the second heating gave better-separated peaks at nearly the same temperatures as in the first heating. After the third cycle, the thermogram became highly reproducible, giving two endothermic peaks at 85.2 and 104.7 °C on heating and a broad exothermic peak at 41.2 °C. Average peak areas (∆H) were +30.7, +28.7, and -68.1 J/g dry chitin, respectively. Though there is approximately 12% discrepancy in ∆H
Figure 2. DSC thermograms of wet β-chitin including reversions between transitions: 1, first heating to 150 °C including a cycle between 90 and 75 °C; 2, cycle between 150 and 65 °C; 3, cooling from 150 to 0 °C and heating to 90 °C; 4, cooling from 90 to 5 °C. All reversions were made with 5 min isothermal holdings. Heating/cooling rate was 20 °C/min.
between the total for endothermic peaks (59.4 J/g of dry chitin) and the exothermic one, they seem to represent the same transition in opposite directions. The discrepancy is possibly caused by temperature dependence of heat capacity, as often experienced in melting and freezing of water in DSC measurements. That the observed DSC peaks show transitions between distinct hydration states was evidenced by the behavior in thermal cycles including reversions between the peaks as follows (Figure 2): Branch 1: The heating was started at 0 °C; heating was stopped at 90 °C, just after the first endothermic peak; the temperature was held there for 5 min and then lowered to 75 °C. As heating was resumed from 75 °C up to 150 °C, the first endothermic peak was not observed and the second endothermic peak appeared at 104 °C just as in the straight heating shown in Figure 1. Branch 2: When the specimen was cooled from 150 to 65 °C and reheated to 150 °C, no peak appeared either on cooling or on heating. Therefore the hydration state of specimen does not seem to change in this treatment. Branch 3: When the specimen was cooled from 150 to 0 °C, a broad exothermic peak between 60 and 0 °C appeared as in Figure 1. Subsequent heating to 90 °C gave the 84 °C endothermic peak as in branch 1. Branch 4: Cooling the specimen from 90 to 0 °C gave a sharp exothermic peak at 72 °C, which had not been observed in cooling from 150 °C. These phenomena clearly show that the observed DSC peaks represent reversible thermal transitions between hydration states of β-chitin. One remarkable feature in Figure 2 (branch 4) is the appearance of sharp exothermic peak at 72 °C on cooling from 90 °C, i.e., before experiencing the 104
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Figure 3. X-ray diffraction patterns of wet β-chitin at varied temperature on heating. Peaks A, B, and C are considered to represent dihydrate, monohydrate, and anhydrous forms of β-chitin, respectively. Table 1. d-Spacings and Indices for Anhydrous and Hydrated Forms of β-Chitin indicesa peak denotation in Figure 3
d-spacing (nm)
A B C D E F G H
1.10 1.03 0.91 0.68 0.52 0.48 0.44 0.42
anhydrous
10.4 Å hydrate
11.6 Å hydrate 002
001 011 020 100 021
002, 010 011 020 200 201 212 h
100 102h 023
a Indices according to Blackwell.2 The index for the same molecular stacking plane differs for different hydration states because of the change in unit cell due to hydration.2 Also these indices do not correspond to the axes in Figure 4 because of the historic change in the crystallographic convention.
°C transition on previous heating. This is a phenomenon not observed in simple heating-cooling cycles between 0 and 150 °C. Since wet β-chitin takes the dihydrate form at room temperature, the most likely interpretation is that the two endothermic peaks on heating represent stepwise dehydration and that the broad exothermic peak on cooling represents the formation of dihydrate from the anhydrous form. The appearance of a sharp exothermic peak on cooling from the putative monohydrate (the state at 90 °C) means that the transition from monohydrate to dihydrate is a rapid process, while that from anhydrous form to monohydrate is a slow process. 3.2. X-ray Diffraction at Varied Temperature. To corroborate the interpretation of the DSC results, we performed X-ray diffraction analysis of wet β-chitin at varied temperatures (Figure 3). All the major peaks could be assigned to the indices described by Blackwell2 as in Table 1. Starting from room temperature, the pattern showed a systematic change with increasing temperature. The most important change appeared in the small angle region; i.e.,
Figure 4. Schematic drawings of anhydrous and hydrated β-chitin crystals. The molecular chains (c axis) lie perpendicular to the drawn plane. The chitin molecules form sheets in the ac plane by stacking. Sheets are stabilized by van der Waals forces between glucopiranoside rings and intermolecular hydrogen bonds between amide groups. Water molecules are intercalated between the sheets reversibly.
the peak at 8° (peak A in Figure 3) in the room-temperature pattern diminished at 60-80 °C and the 8.5° peak (B) became dominant, then the latter diminished at above 100 °C, with the 9.8° peak (C) becoming dominant. The transitions in diffraction pattern are rather gradual, probably because of inhomogeneity of the processes due to larger sample size than in DSC. Still the overall change corresponds well with the process observed by DSC. This series of changes in X-ray diffraction on heating can be interpreted as thermal dehydration of β-chitin as follows. Figure 4 shows schematically the changes in unit cell in hydration of β-chitin. Its crystal structure is characterized by close stacking of the straight and planar poly(N-acetylD-glucosamine) chains, giving rise to the formation of dense sheets defining the ac plane. These sheets are apparently stabilized by the van der Waals forces between the hydrophobic faces of glucopyranoside rings and also by the hydrogen bonds of amide groups in vertical direction.7 In contrast, the binding between the sheets, i.e., the attraction in b direction, is weak because of poor contacts and the lack of hydrogen bonds. This anisotropy is considered to be the cause of ready hydration that takes place by intercalation of water between the ac sheets.5 On the basis of this model the diffraction peaks A-C in Figure 3 have been ascribed to dihydrate, monohydrate, and anhydrous forms of β-chitin.2 Therefore the thermally induced changes in diffraction pattern correspond well with the transitions detected by DSC; i.e., the endothermic peaks at 85.2 and 104.7 °C on heating plausibly correspond to transitions of dihydrate to monohydrate and monohydrate to anhydrous forms, respectively. Reproducibility of the
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transitions in DSC measurements indicates that the parallelchain arrangement and the crystallinity of β-chitin are maintained through the thermal treatments. One material commonly cited as β-chitin is that of squid pen, and there are several reports supporting this identification, including X-ray diffraction pattern of hydrates.6,10 Our DSC test with this material, however, did not give any peak. Since crystallinity of the squid pen chitin is significantly lower than that of Lamellibrachia studied here, it may be the case that the thermal detection of hydration/dehydration requires higher crystalline orders. 4. Conclusion There are several polysaccharides that form crystalline hydrates, including nigeran11 and VH amylose,12 but the hydration of β-chitin seems unique in that the orderly stacked polymeric sheets are readily intercalated with water molecules. This anisotropy is considered to be the cause of the thermally reversible conversion between various hydration states as evidenced here. There are several aspects of the phenomenon to be elucidated, such as accurate gravimetry of hydration/dehydration processes and possible effects of heating/cooling rates on the thermal transitions. The phenomenon is potentially useful as means of characterization of chitin crystals and may provide insights to the interaction of water with crystalline polysaccharides. Also, further studies on the hydrates and other crystallosolvates of β-chitin are expected to lead to novel functional materials.
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Acknowledgment. We thank Professor S. Ohta of The Ocean Research Institute, The University of Tokyo, for providing the Lamellibrachia tube specimens. This research was partly supported by Grant-in-Aid for Scientific Research, No. 10760105 and No. 11760220, the Ministry of Education, Science, Sports and Culture, Japan.
References and Notes (1) (2) (3) (4)
(5) (6) (7) (8) (9) (10) (11) (12)
Rudall, K. M.; Kenchington, W. Biol. ReV. 1973, 49, 597. Blackwell, J. Biopolymers 1969, 7, 281. Minke, R.; Blackwell J. J. Mol. Biol. 1978, 120, 167. Saito, Y.; Okano, T.; Putaux, J. L.; Gaill, F.; Chanzy, H. In AdVances in Chitin Science, SeVenth ICCC; Jacques Andre´ Publisher: Lyon, France, 1997; Vol. II, p 507. Saito, Y.; Okano, T,.; Gaill, F.; Chanzy, H.; Putaux, J. L. Int. J. Biol. Macromolecules 2000, 28, 81. Dweltz, N. E. Biochim. Biophys. Acta 1961, 51, 283. Gardner, K. H.; Blackwell, J. Biopolymers 1975, 14, 1581. Gaill, F.; Persson, J.; Sugiyama, J.; Vuong, R.; Chanzy, H. J. Struct. Biol. 1992, 109, 116. Sugiyama, J.; Boisset, C.; Hashimoto, M.; Watanabe, T. J. Mol. Biol. 1999, 286, 247. Tanner, S.; Chanzy, H.; Vincendon, M.; Roux, J. C.; Gaill, F. Macromolecules 1990, 23, 3576. Taylor, K. J.; Chanzy, H.; Marchessault, R. H. J. Mol. Biol. 1975, 92, 165. Brisson, J.; Chanzy, H.; Winter W. T. Int. J. Biol. Macromol. 1991, 13, 31.
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