Cu(Ⅱ)、Zn(Ⅱ)水溶液的微观结构及其对牛血清白蛋白构象的影响
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摘要
铜和锌是人体必需的微量元素,具有广泛的生物功能。对其毒理作用已经作了很多有意义的研究。在铜和锌对人体的致病过程中,水作为溶剂和维持蛋白质构象的稳定起着至关重要的作用。本论文对Cu~(2+)、Zn~(2+)对水分子团簇结构的影响和其水合离子结构进行了研究,并通过冷冻-解冻的方法改变其结构,进一步研究不同结构Cu~(2+)和Zn~(2+)水合离子和水分子团簇条件下牛血清白蛋白(BSA)构象的变化规律。论文主要包括以下几个部分:
利用粘度法、核磁共振和拉曼光谱等方法,研究了ZnCl_2、AlCl_3、CuCl_2和CuSO4及其阴、阳离子对水分子结构的影响,这些化合物均具有促进水分子缔合,使水团簇加大的作用。阳离子对水结构的缔合促进作用程度为Al_3+>Zn~(2+)>Cu~(2+),Cl-对水缔合的破坏作用大于SO42-。
三蒸水和不同浓度ZnCl_2、AlCl_3、CuCl_2溶液经冷冻处理后,水团簇变小,电导率增加,17O-NMR化学位移和半峰宽降低,这种结构可保持8小时左右。Cu~(2+)和Zn~(2+)水合离子随溶液浓度增加,第一水合层配位结构发生变化。Cu-O配位数介于3～4之间,键长在1.92～1.98 ?之间。随溶液浓度增加,Cu~(2+)原先结合O原子的位置被取代,配位数减小,键长增加;Zn-O配位数介于5～6之间,Zn-O键长在2.03～2.05 ?之间,随溶液浓度增加,水解络合物Zn(OH)n(H2O)m(2-n)所占的比例增大,配位数和键长均减小。
Cu~(2+)和Zn~(2+)水溶液经冷冻-解冻处理后,溶液中自由水分子增多,金属离子水合效应增强,第一水合层配位结构发生明显变化。Cu-O配位数变大,键长变化无规律,热无序度增加;Zn-O配位数和键长均变大。
Cu~(2+)和Zn~(2+)与BSA有强的结合作用,随摩尔比的增加,对BSA氢键的破坏作用增强,同时由于Cu~(2+)和Zn~(2+)对水结构的促进作用,水分子与BSA之间的疏水作用降低,BSA的稳定性降低,α-螺旋结构含量逐渐降低。对5.0×10-6mol·L-1BSA溶液,Cu~(2+)与BSA的摩尔比为5～15,Zn~(2+)与BSA摩尔比为10～15时,出现了由于Cu~(2+)和Zn~(2+)对BSA疏水基团作用的增大,α-螺旋结构增加的中间构象状态。
CuCl_2和Zn(NO3)2溶液经冷冻处理,在解冻后8小时内,引起BSA紫外吸收增色效应、红外光谱酰胺Ⅰ带与酰胺Ⅱ带的位移和CD谱的峰强度变化减弱,对α-螺旋结构的氢键破坏作用降低,α-螺旋结构含量降低的程度变小。冷冻处理三蒸水后溶解BSA和CuCl_2、Zn(NO3)2反应,对BSA的构象影响明显。
As essential microelements, copper and zinc have important biological functions for human body. There had many meaningful studies on their toxicological effects. However, during the process of causing diseases to human body by copper and zinc, water act as important role which is the solvent and maintains the conformation stabilization of proteins. In this thesis, the effect of Cu~(2+)、Zn~(2+) on water cluster structure and the structure of its hydrated ions were studied, furthermore, the structures of water cluster and hydrated ions were changed by freeze-thaw disposing, and the change rule of BSA conformation correlated with different structures of water cluster and hydrated ions were investigated too. The detailed work of this thesis has been set out as follows:
The influences of ZnCl_2、AlCl_3、CuCl_2、CuSO4 and their individual cations and anions on the water structure were studied with viscosity measurement、17O-NMR and Raman spectra. It was proved that all these compounds increased water association and the size of water clusters. The degree of cations increasing effect on water association were Al_3+>Zn~(2+)>Cu~(2+), and Cl- broke more water clusters than SO42-.
After freeze-thaw disposed on the trinal distilled water and different concentrations of ZnCl_2、AlCl_3 and CuCl_2 solutions, the water cluster size became smaller, the conductivity increased, the 17O-NMR chemical shift and half-height widths of peaks decreased obviously, and these structures could hold at least 8 hours.
The first hydrated shell coordination structure of Cu~(2+) and Zn~(2+) hydrated ions changed with the increasing of aqueous solution concentrations. Cu~(2+) had 3～4 oxygen nearest neighbors with the Cu-O distance being 1.92～1.98 ?. With increasing salt concentration, the coordination station of oxygen atom coordinated with Cu~(2+) were replaced which resulted in the coordination number of Cu~(2+) decreased and the Cu-O distance increased in its first coordination shell; the coordination number of Zn-O were 5～6 with the distance being 2.03～2.05 ?, the proportion of hydrolysis complexes Zn(OH)n(H2O)m(2-n) was increased with the aqueous solution concentrations increasing, which induced the Zn-O coordination number and distance minished.
The first coordination shell structure of Cu~(2+) and Zn~(2+) hydrated ions changed obviously after freeze-thaw disposed resulted from the enriching of free water molecular and enhancing of hydrated effects of metal ions. The coordination number of Cu~(2+) and thermal disorder degree increase in first coordination shell, and there was no obvious chang rule of Cu-O distance; the Zn-O coordination number and distance increased in first coordination shell after freeze-thaw treatment.
Cu~(2+) and Zn~(2+) combined with BSA strongly. The breaking effect on hydrogen bonds enhanced with the mole ratio of metal ions and BSA increasing, and for the increasing effect of Cu~(2+) and Zn~(2+)on water association, the hydrophobic effect between H2O and BSA molecules decreased. These two reasons resulted in the change of stability of BSA and decreasing of the amounts ofα-helices. To BSA solution of 5.0×10-6 mol·L-1, there was a intermediate state of the amounts ofα-helices increased for an enhancement in the hydrophobic interactions of BSA molecule with Cu~(2+) and Zn~(2+) when the mole ratio of Cu~(2+) and BSA were 5～15, Zn~(2+) and BSA were 10～15.
It can be conclude that the interaction of CuCl_2 and Zn(NO3)2 solutions on BSA weakened in 8 hours after freeze-thaw disposing on CuCl_2 and Zn(NO3)2 solutions, which resulted in the weakening of UV hyperchromic effect、FTIR amideⅠandⅡbands shift and the change of CD spectra intensity, the decreasing of breaking effect on hydrogen bonds inα-helices of BSA, and the lessening of decreasing degree of theα-helices amounts. The effect on BSA conformation was more obviously when CuCl_2 and Zn(NO3)2 reacted with BSA dissolved in the trinal distilled water after freeze-thaw disposed.
利用粘度法、核磁共振和拉曼光谱等方法,研究了ZnCl_2、AlCl_3、CuCl_2和CuSO4及其阴、阳离子对水分子结构的影响,这些化合物均具有促进水分子缔合,使水团簇加大的作用。阳离子对水结构的缔合促进作用程度为Al_3+>Zn~(2+)>Cu~(2+),Cl-对水缔合的破坏作用大于SO42-。
三蒸水和不同浓度ZnCl_2、AlCl_3、CuCl_2溶液经冷冻处理后,水团簇变小,电导率增加,17O-NMR化学位移和半峰宽降低,这种结构可保持8小时左右。Cu~(2+)和Zn~(2+)水合离子随溶液浓度增加,第一水合层配位结构发生变化。Cu-O配位数介于3～4之间,键长在1.92～1.98 ?之间。随溶液浓度增加,Cu~(2+)原先结合O原子的位置被取代,配位数减小,键长增加;Zn-O配位数介于5～6之间,Zn-O键长在2.03～2.05 ?之间,随溶液浓度增加,水解络合物Zn(OH)n(H2O)m(2-n)所占的比例增大,配位数和键长均减小。
Cu~(2+)和Zn~(2+)水溶液经冷冻-解冻处理后,溶液中自由水分子增多,金属离子水合效应增强,第一水合层配位结构发生明显变化。Cu-O配位数变大,键长变化无规律,热无序度增加;Zn-O配位数和键长均变大。
Cu~(2+)和Zn~(2+)与BSA有强的结合作用,随摩尔比的增加,对BSA氢键的破坏作用增强,同时由于Cu~(2+)和Zn~(2+)对水结构的促进作用,水分子与BSA之间的疏水作用降低,BSA的稳定性降低,α-螺旋结构含量逐渐降低。对5.0×10-6mol·L-1BSA溶液,Cu~(2+)与BSA的摩尔比为5～15,Zn~(2+)与BSA摩尔比为10～15时,出现了由于Cu~(2+)和Zn~(2+)对BSA疏水基团作用的增大,α-螺旋结构增加的中间构象状态。
CuCl_2和Zn(NO3)2溶液经冷冻处理,在解冻后8小时内,引起BSA紫外吸收增色效应、红外光谱酰胺Ⅰ带与酰胺Ⅱ带的位移和CD谱的峰强度变化减弱,对α-螺旋结构的氢键破坏作用降低,α-螺旋结构含量降低的程度变小。冷冻处理三蒸水后溶解BSA和CuCl_2、Zn(NO3)2反应,对BSA的构象影响明显。
As essential microelements, copper and zinc have important biological functions for human body. There had many meaningful studies on their toxicological effects. However, during the process of causing diseases to human body by copper and zinc, water act as important role which is the solvent and maintains the conformation stabilization of proteins. In this thesis, the effect of Cu~(2+)、Zn~(2+) on water cluster structure and the structure of its hydrated ions were studied, furthermore, the structures of water cluster and hydrated ions were changed by freeze-thaw disposing, and the change rule of BSA conformation correlated with different structures of water cluster and hydrated ions were investigated too. The detailed work of this thesis has been set out as follows:
The influences of ZnCl_2、AlCl_3、CuCl_2、CuSO4 and their individual cations and anions on the water structure were studied with viscosity measurement、17O-NMR and Raman spectra. It was proved that all these compounds increased water association and the size of water clusters. The degree of cations increasing effect on water association were Al_3+>Zn~(2+)>Cu~(2+), and Cl- broke more water clusters than SO42-.
After freeze-thaw disposed on the trinal distilled water and different concentrations of ZnCl_2、AlCl_3 and CuCl_2 solutions, the water cluster size became smaller, the conductivity increased, the 17O-NMR chemical shift and half-height widths of peaks decreased obviously, and these structures could hold at least 8 hours.
The first hydrated shell coordination structure of Cu~(2+) and Zn~(2+) hydrated ions changed with the increasing of aqueous solution concentrations. Cu~(2+) had 3～4 oxygen nearest neighbors with the Cu-O distance being 1.92～1.98 ?. With increasing salt concentration, the coordination station of oxygen atom coordinated with Cu~(2+) were replaced which resulted in the coordination number of Cu~(2+) decreased and the Cu-O distance increased in its first coordination shell; the coordination number of Zn-O were 5～6 with the distance being 2.03～2.05 ?, the proportion of hydrolysis complexes Zn(OH)n(H2O)m(2-n) was increased with the aqueous solution concentrations increasing, which induced the Zn-O coordination number and distance minished.
The first coordination shell structure of Cu~(2+) and Zn~(2+) hydrated ions changed obviously after freeze-thaw disposed resulted from the enriching of free water molecular and enhancing of hydrated effects of metal ions. The coordination number of Cu~(2+) and thermal disorder degree increase in first coordination shell, and there was no obvious chang rule of Cu-O distance; the Zn-O coordination number and distance increased in first coordination shell after freeze-thaw treatment.
Cu~(2+) and Zn~(2+) combined with BSA strongly. The breaking effect on hydrogen bonds enhanced with the mole ratio of metal ions and BSA increasing, and for the increasing effect of Cu~(2+) and Zn~(2+)on water association, the hydrophobic effect between H2O and BSA molecules decreased. These two reasons resulted in the change of stability of BSA and decreasing of the amounts ofα-helices. To BSA solution of 5.0×10-6 mol·L-1, there was a intermediate state of the amounts ofα-helices increased for an enhancement in the hydrophobic interactions of BSA molecule with Cu~(2+) and Zn~(2+) when the mole ratio of Cu~(2+) and BSA were 5～15, Zn~(2+) and BSA were 10～15.
It can be conclude that the interaction of CuCl_2 and Zn(NO3)2 solutions on BSA weakened in 8 hours after freeze-thaw disposing on CuCl_2 and Zn(NO3)2 solutions, which resulted in the weakening of UV hyperchromic effect、FTIR amideⅠandⅡbands shift and the change of CD spectra intensity, the decreasing of breaking effect on hydrogen bonds inα-helices of BSA, and the lessening of decreasing degree of theα-helices amounts. The effect on BSA conformation was more obviously when CuCl_2 and Zn(NO3)2 reacted with BSA dissolved in the trinal distilled water after freeze-thaw disposed.
引文
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