【作者】 李红兵；

【导师】 何志敏；

【作者基本信息】 天津大学 ， 化学工程， 2005， 博士

【摘要】 海藻酸钠作为天然生物大分子,由于其立体结构的特殊性,表现出特殊的物理化学性质,具有可生物降解、良好的生物相容性、和生物黏附性等特性,从而广泛地应用于医药和生物技术等领域。本文用热力学、光谱学等方法对海藻酸钠凝胶机理进行了分析。并对海藻酸钠的结构组成和特种海藻酸钠品种的制备进行了探讨。用等温滴定量热法研究了钙锌离子与海藻酸钠相互作用的热力学过程。量热滴定拟合结果表明,Ca²⁺与海藻酸钠相互作用时有两种结合位点,分别对应K₁=1.21E7(L·mol^-1) ,ΔS₁=141.84(J·mol^-1·K^-1) ,ΔH₁=1.89(kJ·mol^-1) ; K₂=2.90E4(L·mol^-1),ΔS₂=79.08(J·mol^-1·K^-1),ΔH₂=^-1.84(kJ·mol^-1)。锌与海藻酸钠相互作用时只有一种结合位点,结合常数K=9472(L·mol^-1) ,熵变ΔS=128.95(J·mol^-1·K^-1),焓变ΔH=15.8(kJ·mol^-1)。钙与海藻酸钠的相互作用比锌与海藻酸钠的相互作用强。扫描电镜照片表明,海藻酸锌凝胶比海藻酸钙凝胶交联点多,空腔比较小,腔与腔之间的隔膜比较多。用圆二色谱对海藻酸钠与钙锌离子相互作用的稳态溶液-凝胶过程进行了研究,结果表明该过程分前后两个阶段,在临界点之前第一阶段内,海藻酸钠与阳离子生成的凝胶配合物结构在线性变化范围内是相同的,同时速度和机理也是一样的。对海藻酸钠与钙锌离子的选择识别和相互作用研究表明,钙离子与海藻酸钠的G、M单元相互作用差别很大,锌离子与G、M单元相互作用差别比较小。锌离子对G、M单元的识别作用不如钙离子。通过四个不同组成的海藻酸钠样品圆二色CD信号随温度变化的谱图,结合X-射线衍射、13C-NMR技术证明海藻酸钠中存在双螺旋结构的功能微区。G段形成的双螺旋功能微区结构,随温度发生有序-无序可逆转变。海藻酸钠与二价阳离子凝胶时会发生膨胀收缩现象。对Cu²⁺、Ca²⁺、Zn²⁺离子来说,膨胀收缩程度的大小,与离子大小,成键电子轨道等离子性质有关。且膨胀和收缩是可逆的。在海藻酸钠与钙离子结合过程中,量热反应的第一类结合位点,对应于离子量对圆二色谱影响的第一阶段线性部分,钙镶嵌于已存在的螺旋蛋盒空腔中,相似于分子内交联,使粘度有稍微下降的趋势。半蛋盒结构以及二聚体聚集体形成过程,对应于量热反应的第二类结合位点,在圆二色CD谱图上,对应于第二阶段的斜率比值比第一阶段小。在这一过程中,以分子间交联为主,体系粘度急剧上升。Cu²⁺、Ca²⁺、Zn²⁺与海藻酸钠相互作用时,体系的焓变、熵变符合等动力学关系,说明三种离子与海藻酸钠的相互作用都是静电离子相互作用。古罗糖醛酸裂解酶（G-lyase）降解poly-G-blocks所得七种寡糖的产率分别为39.5%、22.1%、21.3%、2.2%、2.4%、8.3%与1.2%。结构鉴定结果说明,产物中含有一种二聚糖ΔG（39.5%）;二种三聚糖（ΔGG,22.1%;ΔMG,21.3%）;产物中不含+ΔM,ΔMM或者ΔGM;产物中甘露糖醛酸的含量从9%增加到10.68%,M含量增加了18.7%。根据以上实验结果推测裂解位点位于两个古罗糖醛酸之间（-G-G-）,使得一个古罗糖醛酸变成不饱和糖醛酸位于产物的非还原末端,而另一个古罗糖醛酸位于产物的还原末端。这些结果验证了G-lyase是古罗糖醛酸专一裂解酶。此外从产物含量可知,五聚糖只占3%,六聚糖几乎没有检测到,说明G-lyase酶所能识别的最长链段为五聚糖。用¹³C-NMR结合马尔可夫（贝努利）统计理论对海藻酸钠LVCR的结构进行分析,结果表明LVCR中G单元含量为39.77%。更多还原

【Abstract】 Sodium alginate,a natural biomolecule,shows special physicochemical properties due to steric structure characterity.Being biodegradable,biocompliance and bioadhesive,alginate has being widely used in the biological and pharmaceutical fields.For better application of alginate,it is necessary that we study its sol-gel mechanism with thermodynamic and spectral methods from the physicochemical properties.Simultaneously,alginate composition showed be determined and preparation of its special samples be studied.Thermodynamics of the interaction of calcium and zinc ions with sodium alginate has been studied by isothermal titration Calorimetry.The results showed that there are two sites when calcium ion complexates with alginate, with K₁=1.21E7(L·mol^-1) ,ΔS₁=141.84(J·mol^-1·K^-1) ,ΔH₁=1.89(kJ·mol^-1) ; K₂=2.90E4(L·mol^-1),ΔS₂=79.08(J·mol^-1·K^-1),ΔH₂=^-1.84(kJ·mol^-1).There is only one site when zinc ion interacts with alginate,with the binding affinity K=9472(L·mol^-1),entropyΔS=128.95(J·mol^-1·K^-1),enthalpyΔH=15.8(kJ·mol^-1).The binding affinity of calcium ion complexating with alginate are greater than zinc ion.The scanning electron microscopy（SEM） micrograph showed that there are more linking points ,smaller cavities,and more septa between the cavities in Zn-alginate gel than in Ca-alginate gel.The steadystate interaction research of calcium and zinc ions with alginate during the sol-gel thransition by circular dichroism,show this process includes the former and latter two phases.During the first linear change scale, the structure of the gel-conjugate of alginate-metal ions is homological before the critical point.The rate and mechanism have the similar change.The research of selectivity,recognition and interactions of calcium and zinc ions with alginate, shows that the interactions of calcium ion with guluronic and mannuronic units are more different than that of zinc ion.The selectivity and recogonition of zinc ion for guluronic and mannuronic units are less than calcium ion.The circular dichroism spectra with temperature of four different composition alginate,combined with X-ray diffraction and 13C-NMR spectra technology,showed that there are functional microdomains in alginate. The functional microdomains formed by guluronate units （G）,show order-disorder transitions with temperature.Swelling and shrinking,when alginate gels with divalent cations.For cupric,calcium and zinc ions,the swelling and shrinking degree are related to the cationic properties,such as the ionic radius and bond orbits.Furthermore,swelling and shrinking are reversible.The primary ITC binding sites for alginate interaction with calcium ion correspond to the first linear scale in the CD spectra of intensity variation with calcium-ion concentration.The calcium ions inlay in the“egg-box”cavities,similar with the intramolecular linkage,leading a little decrease for the viscosity.The formation process of the half-egg-box structure and dihelix aggrations ,correspond to the second ITC binding sites.The slope ratio for the second phase are smaller than the first.During this process,the intermolecular linkage are dominating,and the relative viscosity hoiks.Calcium,zinc,cupric ions interact with alginate,their enthalpies and ehtropies show isokinetic relationship,confirming that their interaction mechanisms are the same.Seven oligosaccharides were gained from poly-G-blocks hydrolyzed by guluronate lyase,with yields of 39.5%,22.1%,21.3%,2.2%,2.4%,8.3% and 1.2%,respectively.Structural elucidation showed that only one disaccharide（ΔG,39.5%） and two trisaccharides（ΔGG,22.1%;ΔMG,21.3%） were separated from the mixture.Based on the fact that noΔM,ΔMM orΔGM fractions were separated,we postulated that the hydrolysis mainly occurred between two guluronic acids（-G-G-） making one guluronic acid（G） residue on the reducing end and an unsaturated guluronic acid（Δ） in the nonreducing end.These results proved that the G-lyase was a guluronic acid specific lyase.Furthermore,a very low yield of pentasaccharide（3%） and no trace amount of hexasaccharide indicated that the minimal recognition oligosaccharides of G-lyase should be pentasaccharide.The structure composition of sodium alginate LVCR was analyzed by ¹³C-NMR with Markov（Bernoullian） statistics,the result showed that the amount of G units in alginate LVCR is 39.77%.更多还原