微量量热法研究温敏大分子在溶液中的相变行为
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摘要
本论文中,我们利用微量差示扫描量热法(US-DSC)和压力扰动量热法(PPC)等方法研究了温敏性大分子的相转变行为,包括:聚(N-异丙基丙烯酰胺)(PNIPAM)和聚(N-正丙基丙烯酰胺)(PNNPAM)在水溶液的相转变行为;PNIPAM在聚乙二醇(PEG)构成的拥挤环境中的相转变;溶菌酶在水溶液中的热变性和重复热变性;以及溶菌酶在PEG构成的拥挤环境中的热变性(thermaldenaturation)和重复热变性(thermal redenaturation)。主要结果如下:
(1)我们用US-DSC和PPC法研究了PNIPAM在稀溶液中的聚集和解聚集过程。结果表明,PNIPAM链的聚集和解聚集与温度扫描速率有关,为动力学控制过程。在以往的研究中,已发现PNIPAM的解聚集过程滞后于相应聚集过程,在本工作中我们证实这是由于PNIPAM在塌缩状态时形成的分子内和分子间附加氢键造成的。较低的降温速率下US-DSC曲线出现了两个放热峰,表明解聚集分为两个过程,即附加氢键断裂和聚集体的解聚集。PPC研究表明降温时PNIPAM链的溶剂接触表面积比加热时的小,进一步证实PNIPAM聚集体的解聚集由以上两个过程组成.
(2)我们用US-DSC法研究了PNIPAM链在稀溶液和亚浓溶液中的聚集和解聚集过程。结果表明,当溶液浓度低于临界交叠浓度(C~*)时,加热过程中的转变温度(T_p)和转变焓(ΔH)均随浓度的增加而下降;当溶液为亚浓溶液时,T_p信和ΔH与浓度变化无关。可见,在亚浓溶液中高分子链主要发生链间聚集,在稀溶液中则链间聚集和链内塌缩(折叠)共存。随着浓度的降低,链内塌缩占的比重增加,当外推浓度趋于零时,我们得到了PNIPAM从无规线团到塌缩球转变(coil-to-globule transition)的焓变AOS。AHs高于链间聚集的焓变,说明高分于单链折叠不同于宏观相变。(3)我们用US-DSC法研究了PEG形成的拥剂环境下的PNIPAM的塌缩与聚集过程。结果表明,由于PEG的水合作用导致了r信随PEG的浓度(CPEG)增加而下降。在一个加热.降温循环中,r信在降温过程中产生“滞后”。随着CPEO增加,较低分子量的PEG使该滞后加剧,而加入较长的PEG链则导致滞后变弱。这一现象表明,较长的PEG对PNIPAM链的链间聚集起抑制作用。当C_(PEG)高于其C~*时,“滞后”与C_(PEG)的变化无关,这可能是因为PEG链形成了瞬时网络,一些PNIPAM链被限制在网孔中,其塌缩和聚集范围受到限制,形成的附加氢键有限。此外,随着PEG浓度增加,转变过程变宽表明这种网状结构不均一,具有不同尺寸的网孔。而ΔH/随PEG浓度增加而变小,说明PEG削弱了PNIPAM链的链间聚集作用。ΔH在加热与降温过程中接近,进一步证实了PEG分子对PNIPAM的链间聚集有阻碍作用。
(4)我们用US-DSC法研究了加热/降温速率和浓度对PNNPAM在水溶液中的塌缩与聚集的影响。结果表明,在加热过程中,PNNPAM在-24℃发生了LCST转变。ΔH随着加热速率和浓度的增加而变大。特别是在降温过程中T_p与加热时的基本一致,表明聚集过程中没有形成附加氢键。此外,在降温过程中,出现了多个转变过程,表明PNNPAM解聚集经历了多步水合过程,最终成为线团状态。
(5)我们用US-DSC法研究了研究了浓度、离子强度和pH等因素对溶菌酶热变性和重复热变性的影响。结果表明,随着溶菌酶浓度的增加,T_p增加,并且位于低温侧的吸热峰变强,表明浓度的变大促进了变性过程中溶菌酶的聚集.在热变性和重复热变性过程中,T-p均随加热速率变大而升高,表明在溶菌酶的热变性过程中存在聚集现象,较慢的加热速率有利于其聚集。随着离子强度增加,第一次热变性时的T_p下降,并且峰形变宽。这表明较多的NaCl可破坏溶菌酶分子周围的水合环境而使其变性。当NaCl浓度高于0.6 mol/L时,第二次热变性时的溶菌酶完全失去活性。另外,T_p随pH变大而升高。这是由于随着pH值的变大,溶菌酶的α-螺旋含量减小,结构变得更加松散,热稳定性提高。
(6)我们用US-DSC法研究了溶菌酶在EG和PEG构成的拥挤环境中的热变性与重复热变性。结果表明,EG的加入使溶菌酶变性温度增加,热稳定性提高。当M_(w,PEG)＜1000 g/mol时,T_p随分子量增加而有所下降,并且吸热峰强度也有所减弱,表明较短的PEG分子链容易引起溶菌酶的聚集。当1000 g/mol＜M_(w,PEG)＜2000 g/tool时,T_p随PEG的浓度增加而下降,表明较低分子量的PEG有利于加速溶菌酶的聚集。当6000＞M_(w,PEG)≥2000 g/mol时,T_p几乎不随PEG的浓度变化而变化。此外,ΔH随PEG浓度增加而变小。这是由于加入的PEG抑制了溶菌酶的水合作用导致了ΔH变小。溶菌酶热变性时的协同单元数(n)随PEG浓度增加而下降,进一步证实了PEG的加入促进了溶菌酶分子间的相互作用。
The thesis deals with the phase transition of poly(N-isopropylacrylamide) (PNIPAM) in dilute and semidilute aqueous solutions as well as in in aqueous solutions with polyethylene glycol(PEG) as the crowding agent,association and dissolution of linear poly(N-n-propylacrylamide)(PNNPAM) chains in water,the thermal denaturation and re-denaturation behaviors of lysozyme in aqueous solutions and aqueous solutions with polyethylene glycol(PEG) as the crowding agent.Such behaviors of the polymers have been investigated by use of ultra sensitive differential scanning calorimetry(US-DSC) and pressure perturbation calorimetry(PPC).The results are as follows.
1.Aggregation and dissolution of PNIPAM in water were investigated using an US-DSC and a PPC.US-DSC reveals that both the aggregation and dissolution of PNIPAM chains are greatly dependent on the scanning rate,indicating that the processes are kinetically controlled.The hysteresis in the dissolution process was found to have a nonequilibrium nature,which is thought to be related to the additional hydrogen bondings formed in the collapsed state of PNIPAM chains.A birnodal appearing in the cooling process at a slow scanning rate indicates the dissolution involves two different processes,i.e.,the disruption of additional hydrogen bondings and the dissolution of the collapsed chains.PPC reveals that the solvent accessible surface area of PNIPAM chains in the cooling process is smaller than that in the heating process,which further indicates the dissolution of the PNIPAM aggregates involves such two processes.
2.The concentration(C) effect on association and dissolution of linear PNIPAM chains in dilute and semidilute aqueous solutions was investigated by using US-DSC. When C is below the overlap concentration(C~*),both the phase-transition temperature(T_p) and enthalpy change(△H) in the heating process decrease as the solution becomes more dilute,while T_p and△H become independent of C in the semidilute regime(C＞C~*).By extrapolating concentration to zero,we have obtained the phase transition temperature(T_s) and enthalpy change(△H_s) of the single chain folding.△H_s is higher than that for a phase transition involving intrachain collapse and interchain aggregation,indicating that a single chain folding can not be taken to be a macroscopic phase transition.
3.Collapse and aggregation of PNIPAM chains in aqueous solutions with PEG as the crowding agent have been investigated by using US-DSC.For any PEG with a certain molecular weight,the transition temperature of PNIPAM decreases with PEG concentration(C_(PEG)) due to the complexation between PEG chains and water molecules.A hysteresis has been observed in one heating-and-cooling cycle.As C_(pEG) increases,short PEG chains lead the hysteresis to enlarge,whereas longer PEG chains result in a decrease in the hysteresis.The facts indicate that longer PEG chains suppress the interchain aggregation of PNIPAM chains.When C_(PEG) is above the overlap concentration(C~*),the hysteresis is almost independent of PEG concentration, suggesting that PEG chains form a transient network which locates PNIPAM chains in a number of pores.The widening of the transition with increasing C_(PEG) at C_(PEG)＞C~* indicates that the network is inhomogeneous.The fact that△H decreases with C_(PEG) further indicates that PEG chains reduce the interchain aggregation of PNIPAM chains.The same△H in heating and cooling processes demonstrates that PNIPAM chains form small-scale aggregates in the presence of PEG.
4.The concentration(C) and heating/cooling effects on association and dissolution of linear PNNPAM chains in water were investigated by using US-DSC. In the heating process,PNNPAM chains undergo a LCST transition at～24℃.△H increases with increasing heating rate and concentration.The highest T_p in the cooling process is close to that in the heating process,indicating no additional hydrogen bondings.The multiple-transition observed in the cooling process indicates that the dissolution of aggregates undergoes multiple hydration processes.
5.The effects of lysozyme concentration,ion strength,pH,etc.on the thermal denaturation and re-denaturation behaviors of lysozyme were investigated by using US-DSC.The results reveal that T_p increases with the increasing lysozyme concentration.A new endothermic peak located at lower temperature is observed in the re-denaturation process,which indicates that the increasing concentration induces the association of lysozyme in the heating process.T_p of both the thermal denaturation and re-denaturation processes increase with the inceasing heating rate,suggesting the association behaviors exist in the thermal denaturation process.A slower heating rate promotes the association.As the ion strength increases,T_p in the first heating times decreases and the endothermic peak becomes wide,which is caused by the disruption of the hydration around lysozyme.The excessive amounts of NaCl in solution act as the denaturant in the thermal denaturation of lysozyme.The activity of lysozyme disappears in the second heating times when C_(NaCl)＞0.6 mol/L.T_p increases with the increasing pH values.Tp increases from 51.9 to 70.09℃as pH increases from 2.0 to 3.0,which is caused by the decreasing content ofα-helix with increasing pH.
6.The thermal denaturation and re-denaturation of lysozyme solutions with PEG as the crowding agent have been investigated by using US-DSC.Ethyl glycol(EG) in lysozyme solution increases T_p.When M_(w,PEG)＜1000 g/mol,Tp has a small decreases with increasing molecular weight of PEG and the enderthermic peak weaks,which indicates that the shorter PEG chains are easy to induce the association of lysozyme. T_p increases with increasing molecular weight of PEG when M_(w,PEG)＞1000 g/mol.T_p is independent of the change of C_(PEG) when 6000＞M_(w,PEG)＞2000 g/mol.On the other hand,△H decreases with increasing C _(PEG),which is caused the restriction of the hydration of lysozyme by adding PEG.More PEG chains in solution decreases the hydration of lysozyme,△H thus decreases.The number of cooperative unit(n) decreases with increasing C_(PEG),which further confirms that PEG can promote the association of lysozyme.
(1)我们用US-DSC和PPC法研究了PNIPAM在稀溶液中的聚集和解聚集过程。结果表明,PNIPAM链的聚集和解聚集与温度扫描速率有关,为动力学控制过程。在以往的研究中,已发现PNIPAM的解聚集过程滞后于相应聚集过程,在本工作中我们证实这是由于PNIPAM在塌缩状态时形成的分子内和分子间附加氢键造成的。较低的降温速率下US-DSC曲线出现了两个放热峰,表明解聚集分为两个过程,即附加氢键断裂和聚集体的解聚集。PPC研究表明降温时PNIPAM链的溶剂接触表面积比加热时的小,进一步证实PNIPAM聚集体的解聚集由以上两个过程组成.
(2)我们用US-DSC法研究了PNIPAM链在稀溶液和亚浓溶液中的聚集和解聚集过程。结果表明,当溶液浓度低于临界交叠浓度(C~*)时,加热过程中的转变温度(T_p)和转变焓(ΔH)均随浓度的增加而下降;当溶液为亚浓溶液时,T_p信和ΔH与浓度变化无关。可见,在亚浓溶液中高分子链主要发生链间聚集,在稀溶液中则链间聚集和链内塌缩(折叠)共存。随着浓度的降低,链内塌缩占的比重增加,当外推浓度趋于零时,我们得到了PNIPAM从无规线团到塌缩球转变(coil-to-globule transition)的焓变AOS。AHs高于链间聚集的焓变,说明高分于单链折叠不同于宏观相变。(3)我们用US-DSC法研究了PEG形成的拥剂环境下的PNIPAM的塌缩与聚集过程。结果表明,由于PEG的水合作用导致了r信随PEG的浓度(CPEG)增加而下降。在一个加热.降温循环中,r信在降温过程中产生“滞后”。随着CPEO增加,较低分子量的PEG使该滞后加剧,而加入较长的PEG链则导致滞后变弱。这一现象表明,较长的PEG对PNIPAM链的链间聚集起抑制作用。当C_(PEG)高于其C~*时,“滞后”与C_(PEG)的变化无关,这可能是因为PEG链形成了瞬时网络,一些PNIPAM链被限制在网孔中,其塌缩和聚集范围受到限制,形成的附加氢键有限。此外,随着PEG浓度增加,转变过程变宽表明这种网状结构不均一,具有不同尺寸的网孔。而ΔH/随PEG浓度增加而变小,说明PEG削弱了PNIPAM链的链间聚集作用。ΔH在加热与降温过程中接近,进一步证实了PEG分子对PNIPAM的链间聚集有阻碍作用。
(4)我们用US-DSC法研究了加热/降温速率和浓度对PNNPAM在水溶液中的塌缩与聚集的影响。结果表明,在加热过程中,PNNPAM在-24℃发生了LCST转变。ΔH随着加热速率和浓度的增加而变大。特别是在降温过程中T_p与加热时的基本一致,表明聚集过程中没有形成附加氢键。此外,在降温过程中,出现了多个转变过程,表明PNNPAM解聚集经历了多步水合过程,最终成为线团状态。
(5)我们用US-DSC法研究了研究了浓度、离子强度和pH等因素对溶菌酶热变性和重复热变性的影响。结果表明,随着溶菌酶浓度的增加,T_p增加,并且位于低温侧的吸热峰变强,表明浓度的变大促进了变性过程中溶菌酶的聚集.在热变性和重复热变性过程中,T-p均随加热速率变大而升高,表明在溶菌酶的热变性过程中存在聚集现象,较慢的加热速率有利于其聚集。随着离子强度增加,第一次热变性时的T_p下降,并且峰形变宽。这表明较多的NaCl可破坏溶菌酶分子周围的水合环境而使其变性。当NaCl浓度高于0.6 mol/L时,第二次热变性时的溶菌酶完全失去活性。另外,T_p随pH变大而升高。这是由于随着pH值的变大,溶菌酶的α-螺旋含量减小,结构变得更加松散,热稳定性提高。
(6)我们用US-DSC法研究了溶菌酶在EG和PEG构成的拥挤环境中的热变性与重复热变性。结果表明,EG的加入使溶菌酶变性温度增加,热稳定性提高。当M_(w,PEG)＜1000 g/mol时,T_p随分子量增加而有所下降,并且吸热峰强度也有所减弱,表明较短的PEG分子链容易引起溶菌酶的聚集。当1000 g/mol＜M_(w,PEG)＜2000 g/tool时,T_p随PEG的浓度增加而下降,表明较低分子量的PEG有利于加速溶菌酶的聚集。当6000＞M_(w,PEG)≥2000 g/mol时,T_p几乎不随PEG的浓度变化而变化。此外,ΔH随PEG浓度增加而变小。这是由于加入的PEG抑制了溶菌酶的水合作用导致了ΔH变小。溶菌酶热变性时的协同单元数(n)随PEG浓度增加而下降,进一步证实了PEG的加入促进了溶菌酶分子间的相互作用。
The thesis deals with the phase transition of poly(N-isopropylacrylamide) (PNIPAM) in dilute and semidilute aqueous solutions as well as in in aqueous solutions with polyethylene glycol(PEG) as the crowding agent,association and dissolution of linear poly(N-n-propylacrylamide)(PNNPAM) chains in water,the thermal denaturation and re-denaturation behaviors of lysozyme in aqueous solutions and aqueous solutions with polyethylene glycol(PEG) as the crowding agent.Such behaviors of the polymers have been investigated by use of ultra sensitive differential scanning calorimetry(US-DSC) and pressure perturbation calorimetry(PPC).The results are as follows.
1.Aggregation and dissolution of PNIPAM in water were investigated using an US-DSC and a PPC.US-DSC reveals that both the aggregation and dissolution of PNIPAM chains are greatly dependent on the scanning rate,indicating that the processes are kinetically controlled.The hysteresis in the dissolution process was found to have a nonequilibrium nature,which is thought to be related to the additional hydrogen bondings formed in the collapsed state of PNIPAM chains.A birnodal appearing in the cooling process at a slow scanning rate indicates the dissolution involves two different processes,i.e.,the disruption of additional hydrogen bondings and the dissolution of the collapsed chains.PPC reveals that the solvent accessible surface area of PNIPAM chains in the cooling process is smaller than that in the heating process,which further indicates the dissolution of the PNIPAM aggregates involves such two processes.
2.The concentration(C) effect on association and dissolution of linear PNIPAM chains in dilute and semidilute aqueous solutions was investigated by using US-DSC. When C is below the overlap concentration(C~*),both the phase-transition temperature(T_p) and enthalpy change(△H) in the heating process decrease as the solution becomes more dilute,while T_p and△H become independent of C in the semidilute regime(C＞C~*).By extrapolating concentration to zero,we have obtained the phase transition temperature(T_s) and enthalpy change(△H_s) of the single chain folding.△H_s is higher than that for a phase transition involving intrachain collapse and interchain aggregation,indicating that a single chain folding can not be taken to be a macroscopic phase transition.
3.Collapse and aggregation of PNIPAM chains in aqueous solutions with PEG as the crowding agent have been investigated by using US-DSC.For any PEG with a certain molecular weight,the transition temperature of PNIPAM decreases with PEG concentration(C_(PEG)) due to the complexation between PEG chains and water molecules.A hysteresis has been observed in one heating-and-cooling cycle.As C_(pEG) increases,short PEG chains lead the hysteresis to enlarge,whereas longer PEG chains result in a decrease in the hysteresis.The facts indicate that longer PEG chains suppress the interchain aggregation of PNIPAM chains.When C_(PEG) is above the overlap concentration(C~*),the hysteresis is almost independent of PEG concentration, suggesting that PEG chains form a transient network which locates PNIPAM chains in a number of pores.The widening of the transition with increasing C_(PEG) at C_(PEG)＞C~* indicates that the network is inhomogeneous.The fact that△H decreases with C_(PEG) further indicates that PEG chains reduce the interchain aggregation of PNIPAM chains.The same△H in heating and cooling processes demonstrates that PNIPAM chains form small-scale aggregates in the presence of PEG.
4.The concentration(C) and heating/cooling effects on association and dissolution of linear PNNPAM chains in water were investigated by using US-DSC. In the heating process,PNNPAM chains undergo a LCST transition at～24℃.△H increases with increasing heating rate and concentration.The highest T_p in the cooling process is close to that in the heating process,indicating no additional hydrogen bondings.The multiple-transition observed in the cooling process indicates that the dissolution of aggregates undergoes multiple hydration processes.
5.The effects of lysozyme concentration,ion strength,pH,etc.on the thermal denaturation and re-denaturation behaviors of lysozyme were investigated by using US-DSC.The results reveal that T_p increases with the increasing lysozyme concentration.A new endothermic peak located at lower temperature is observed in the re-denaturation process,which indicates that the increasing concentration induces the association of lysozyme in the heating process.T_p of both the thermal denaturation and re-denaturation processes increase with the inceasing heating rate,suggesting the association behaviors exist in the thermal denaturation process.A slower heating rate promotes the association.As the ion strength increases,T_p in the first heating times decreases and the endothermic peak becomes wide,which is caused by the disruption of the hydration around lysozyme.The excessive amounts of NaCl in solution act as the denaturant in the thermal denaturation of lysozyme.The activity of lysozyme disappears in the second heating times when C_(NaCl)＞0.6 mol/L.T_p increases with the increasing pH values.Tp increases from 51.9 to 70.09℃as pH increases from 2.0 to 3.0,which is caused by the decreasing content ofα-helix with increasing pH.
6.The thermal denaturation and re-denaturation of lysozyme solutions with PEG as the crowding agent have been investigated by using US-DSC.Ethyl glycol(EG) in lysozyme solution increases T_p.When M_(w,PEG)＜1000 g/mol,Tp has a small decreases with increasing molecular weight of PEG and the enderthermic peak weaks,which indicates that the shorter PEG chains are easy to induce the association of lysozyme. T_p increases with increasing molecular weight of PEG when M_(w,PEG)＞1000 g/mol.T_p is independent of the change of C_(PEG) when 6000＞M_(w,PEG)＞2000 g/mol.On the other hand,△H decreases with increasing C _(PEG),which is caused the restriction of the hydration of lysozyme by adding PEG.More PEG chains in solution decreases the hydration of lysozyme,△H thus decreases.The number of cooperative unit(n) decreases with increasing C_(PEG),which further confirms that PEG can promote the association of lysozyme.
引文
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