脉冲电场对氨基酸的极化影响及其制备蛋白质纳米管研究
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摘要
本论文通过傅里叶变换衰减全反射红外光谱（Attenuated Total internalReflectance Fourier Transform Infrared spectroscopy，简称ATR-FTIR）、核磁共振氢谱（Proton Nuclear Magnetic Resonance Spectra，简称HNMR）、圆二色谱（CircularDichroism，简称CD）和高效液相色谱（High Performance Liquid Chromatography，简称HPLC）等手段从点到线，从线到面依次分析色氨酸、组氨酸、蛋氨酸、谷胱甘肽（3肽）、合成16肽和聚赖氨酸在脉冲电场（Pulsed Electric Field，简称PEF）处理条件下的红外吸收规律和H原子能量振动规律，进而研究PEF对-乳球蛋白，卵清蛋白的分子键红外吸收和分子微观结构影响规律。并在此基础上，发现脉冲电场可以作为制备卵清蛋白纳米管的新的加工手段和方法，并采用透射电子显微镜（TransmissionElectron Microscope，简称TEM）分析和检测由脉冲电场制备的卵清蛋白纳米管的形貌特征。具体结果如下：
1，探索PEF对单一氨基酸的极化影响规律，结果如下：
（1）随着脉冲处理时间的延长（7.2ms到36ms），PEF诱使组氨酸、色氨酸、蛋氨酸分子中H-O-H伸缩振动，CH2反对称弯曲振动以及NH2剪刀弯曲振动呈现指数增强（Y=a ebx），而C-N伸缩振动和N-H弯曲振动出现指数（Y=a ln(bx)）减弱。同时，脉冲电场处理（电脉冲时间36ms）诱导组氨酸、蛋氨酸、色氨酸的1400cm-1附近峰消失，说明分子中C-O-O-H的弯曲和伸缩振动相对减弱。对应地，脉冲处理诱使3个单一氨基酸的HNMR图谱中所有H原子的分子缔合呈现指数（Y=a ebx）增强。同时，脉冲电场输入时间的增加诱导这3个单一氨基酸溶液电导率和pH值都略有增加。总结地，脉冲电场输入能量和单一氨基酸分子极化呈指数关系：Y=a ebx，即单一氨基酸分子以指数形式（Y=a ebx）接受电脉冲能量。
（2）对于组氨酸分子，当脉冲处理时间在7.2-28.8ms之间时，PEF对其的结构影响较小。然而较长时间（36ms）的PEF处理诱使组氨酸分子结构发生较大改变。推测36ms的PEF处理使得组氨酸聚集。
（3）对于蛋氨酸分子，FTIR和HNMR图谱均表明：PEF诱导蛋氨酸分子中S原子的极性发生变化，即脉冲电场引起S原子自旋耦合，S原子电子云发生变化。
（4）对于色氨酸分子，较短时间（7.2ms和14.4ms）的脉冲处理使得色氨酸分子活性增加，色氨酸吲哚环上的N基团极化，分子自旋耦合增强，即吲哚环中的N原子电子层发生变化。当较长时间（28.8ms和36ms）的脉冲处理使得色氨酸HNMR图谱裂分消失，色氨酸分子吲哚环上的电子云分布趋向稳定。
2，研究了脉冲电场对3肽，合成16肽，聚赖氨酸，-乳球蛋白的极化规律，结果如下：
（1）脉冲电场对氧化性谷胱甘肽分子结构的影响与脉冲电场对单一氨基酸的影响规律相似：氧化性谷胱甘肽分子结构变化和氢键缔合增强与电脉冲输入能量呈指数关系：(y=a ebx)。同时，脉冲电场对氧化性谷胱甘肽分子结构的影响和S原子关系密切。HNMR图谱也表明，脉冲电场诱导氧化性谷胱甘肽分子中S-S浓度呈对数减小，即PEF展开了氧化性谷胱甘肽分子中的二硫键。说明PEF诱导了S原子电子云分布发生变化，与PEF对蛋氨酸分子中S原子的影响相似。
（2）对于聚赖氨酸分子，较短时间（小于50s）脉冲电场诱导聚赖氨酸分子聚集，且分子聚集呈现线性，分子内的H-O-H伸缩振动，NH2剪刀弯曲振动和CH2反对称弯曲振动减小，C-N伸缩振动和N-H弯曲振动增加。然而较高脉冲能量的输入（脉冲时间从75s－175s）使得聚赖氨酸分子变化不明显。结果表明，脉冲电场引起聚赖氨酸分子聚集。
（3）对于合成16肽，较短电脉冲处理使得合成16肽分子打开，脉冲电场诱导合成16肽中H-O-H伸缩振动，NH2剪刀弯曲振动，C-N伸缩振动，N-H弯曲振动，以及CH2反对称伸缩振动呈现(y=ax2+bx+c)趋势，当脉冲电场处理时间为480s时，FTIR吸收强度都达到最小，即合成16肽分子展开程度最大。当电脉冲时间继续延长（600s），合成16肽分子重新聚集。同时，脉冲电场处理(120s)诱使合成16肽的HNMR图谱中1.18ppm位置的吸收峰消失，而在1.46ppm，1.82ppm和2.70ppm位置各出现一个新峰，说明合成16肽分子的展开，且分子中二硫键展开。结合PEF对谷胱甘肽分子结构中二硫键的影响，进一步验证了脉冲电场对蛋白质分子结构的展开与二硫键的展开有重要联系。
（4）对于-乳球蛋白，随着脉冲电场处理时间的延长，脉冲电场诱导-乳球蛋白分子中H-O-H伸缩振动，NH2剪刀弯曲振动和CH2反对称弯曲振动先减小而后增加(y=ax2+bx+c)，而C-N伸缩振动和N-H弯曲振动出现先增大而后减小的趋势(y=ax2+bx+c)，进一步验证了脉冲电场诱导-乳球蛋白分子先打开（小于100s）而后聚集（大于100s）。PEF对-乳球蛋白结构的影响和其对合成16肽的结构影响相似。
3，研究了脉冲电场对卵清蛋白分子结构的影响规律和脉冲电场制备卵清蛋白纳米管的机理探索，结果如下：
（1）脉冲电场输入能量和卵清蛋白的二级结构展开存在线性关系，卵清蛋白二级结构的展开是一个由三级结构到二级结构逐层逐步的过程。且在相同脉冲能量输入的情况下，蛋白质分子结构的展开对高电场强度的脉冲能量激发更敏感，而不是较长时间PEF处理的脉冲能量积累。相对-折叠结构易受热效应的影响，而-螺旋结构则更容易接受电脉冲作用，因为PEF对-螺旋结构中偶极距影响较大。
（2）脉冲电场和电导率对卵清蛋白二级结构的协同影响也呈线性关系。对于相同的PEF处理条件，电导率越高，即Ca2+浓度越高，蛋白质结构改变也越大。对于电导率为302S/cm的溶液接收脉冲电场处理3375s，和电导率为403S/cm的溶液接收脉冲电场处理3750s，以及电导率为516S/cm的溶液接收脉冲电场处理2250s和3000s，卵清蛋白溶液中都有蛋白质纳米管生成。特别地，当电导率为516μS/cm和606μS/cm的溶液接收3000s的PEF处理时，蛋白质纳米管形状清晰规范，纳米管长度在10m左右，外径在200nm左右，内径在30-50nm之间。当脉冲电场处理时间较长（4500s）和溶液电导率较高（816μS/cm）时，卵清蛋白分子聚集。结果说明，卵清蛋白纳米管的形成与脉冲电场参数和Ca2+浓度有关。
（3）对于添加不同摩尔比金属离子的卵清蛋白溶液，脉冲电场输入的脉冲能量和蛋白质分子的展开程度也成线性关系：[q]obs=a·Q+b。在脉冲电场处理条件下，蛋白质纳米管的形成不仅和金属离子的浓度有关，也和金属离子的带电荷数，金属离子的电子层数有关。一价金属离子（Na+）和三价金属离子（Fe3+）对蛋白质纳米管形成的作用不大。对于相同带电荷数的Mg2+，Zn2+，Ba2+，Mn2+，Cu2+和Ca2+而言，Cu2+，Mn2+，Ca2+，Zn2+更容易形成结构清晰的蛋白质纳米管，且它们都由3层电子层组成。Ba2+促使形成的是蛋白质纳米纤维，可能因为拥有5层电子层的原故。相对而言，Mg2+对蛋白质纳米管的形成影响不大，可能因为只有2层电子层。
（4）在脉冲电场处理条件下，对于添加Cu2+，Mn2+，Ca2+，Zn2+的卵清蛋白溶液，蛋白质纳米管的形成是一个递进过程。最初金属离子和蛋白质螯合形成纳米颗粒核，然后在纳米颗粒核的基础上，在脉冲电场的驱动下，蛋白质分子自组装成纳米线，继而2条或几条蛋白质纳米线自组装成蛋白质纳米管。所形成的蛋白质纳米管形状：精致规范，外径在200－300nm之间，内径在20－100nm之间，管长20m左右。当继续施加电脉冲能量时，蛋白质纳米管开始聚集，蛋白质分子变性。蛋白质分子聚集意味着蛋白质分子的变性，但蛋白质纳米管的形成并不意味着蛋白质分子变性，而是蛋白质分子结构改变后的最稳定分子状态，对应分子自由能最小。
（5）脉冲电场和pH协同作用不能形成蛋白质纳米管，然而PEF诱使pH12的卵清蛋白溶液中有蛋白质纳米纤维生成，再次证明蛋白质纳米管的形成需要金属离子作为桥梁。
The effect of pulsed electric fields (PEF) treatments on the molecular structurepolarization of histidine, methionine, tryptophan, Oxidative glutathione (GSSG), synthesized16-peptide and polylysine were investigated in this paper using Attenuated Total InternalReflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR), Proton Nuclear MagneticResonance Spectra (HNMR), Circular Dichroism (CD) and High Performance LiquidChromatography (HPLC). Accordingly, a new application of PEF as a novel technology on thepreparation of ovalbumin nanotubes was developed. The results were listed as followings:
1. Firstly, the effect of PEF treatment on the structure of single amino acid, such ashistidine, tryptophan and methionine, were investigated by FTIR and NMR. When the pulsedelectric time increased from7.2ms to36ms, the PEF treatment induced all stretching vibrationof H-O-H, anti-symmetric bending vibration of CH2and shear-bending vibration of NH2inFTIR spectra increased expontentially. However, the stretching vibration of C-N and bendingvibration of N-H decreased expontentially. When the longest PEF treatment time was applied(36ms), the peak at around1400cm-1disappreared which implied a significant decrease in thebending and stretching vibration of C-O-O-H. While in the HNMR spectra, it was observedthat all peaks shifted toward the lower wavenumber. It was also observed that PEF treatmentsresulted in a little increase in the conductivity and pH value of these three amino acids solution.
1) Especially as for the histidine, when the pulse time increased from7.2ms to28.8ms,the PEF treatment has little influence on its molecular structure. However, when the electricpulse treatment time increased to36ms, its influrence became significant. PEF treatment mayinduced a aggregation of histidine.2) As for the methionine, it was observed from both FTIRand HNMR spectra that PEF induced significant polarization of S atom.3) As for thetryptophan, when the electric pulse time increased to7.2ms and14.4ms, PEF induced thepolarization of N in the indole ring.
2. Thereafter, the effect of PEF on the oxidative glutathione (GSSG) was explored. FTIRanalysis demonstrated that an expontential relationship between the electric pulse energy and the change degree of molecular structure of oxidative glutathione (GSSG) was observed. WhileHNMR spectra implied that the S-S bonds in GSSG unfolded, which can be used to explain themechanism of the effect of PEF on Met.
3. The effect of PEF treatment on the structure of polylysine was inverstigated. Resutlsshowed that the shorter time (50s) PEF treatment induced the linear aggragation ofpolylysine. All peaks in the FTIR spetra about the stretching vibration of H-O-H, anti-symmetric bending vibration of CH2, the shear-bending vibration of NH2, the stretchingvibration of C-N and bending vibration of N-H presented an increase trend. However, when thetreatment time increased from75s to175s, the effect of PEF has little influence onpolylysine.
4. Following, the effect of PEF treatment on the structure of synthetic16peptide wasstudied. Results showed that the shorter PEF treatment time induced its unfolding as well.When the treatment time increased to480s, the most significant unfolding of16peptidestructure was observed. However, when the treatment time further increased to600s,apparently aggregation was observed. Similarly, HNMR spectra demonstrated the unfolding ofS-S bond among16peptide.
5. Furthermore,-lactoglobulin was chosen as the reoresentive to identify the effect ofPEF treatment on the structure of real protein. Results from FTIR analysis showed that with theincrease of PEF treatment time from25s to125s, the stretching vibration of H-O-H, anti-symmetric bending vibration of CH2, the shear-bending vibration of NH2, the stretchingvibration of C-N and bending vibration of N-H changed correspondingly.w When the electricpulse time continucely increased, the FTIR intensity of these above group decreased. Resultssuggested that PEF induced firstly unfolding and secondarily refolding of-lactoglobulin.
6. Finally, the effect of PEF on the molecular structure of ovalbumin and the mechanismof preparation ovalbumin nanotubes were studied.
1) Modulating the input energy via changing pulse intensity and treatment time, astepwise linear unfolding of secondary and tertiary structure of ovalbumin was observed. It isexplored that such change is more liable to electric pulse excitation rather than pulse energy accumulation, and the unfolding degree of tertiary structure was more significant than that ofsecondary structure. Compared with the-sheet was liable to the heat effective, the-helixwas more subjected to the electric pulse treatment due to its dipole moment structure.
2) A linear relationship between electric pulse energy and unfolding structure ofovalbumin were also observed. Under the same PEF treatment condition, the higherconductivity with higher Ca2+concentration, the more changes on the secondary structure ofovalbumin were obtained. It was found that when the sample was treated at the conductivity of302μS/cm or403μS/cm for3375s, or at516μS/cm for2250s, the formation ofovalbuminna notubes were observed. Especially for the PEF treatment with conductivity of516μS/cm or606μS/cm for3000s, the delicate nanotube were observed with the size of10m length×200nm external diameter×30-50nm inner diameter. However, under moresevere condition such as816μS/cm for4500s, the ovalbumin aggregated.
3) For the ovalbumin solution with difference metal ions concentration, the linearunfolding of secondary structure with increasing electric pulse energy was observed. At thesame time, the formation of nanotubes was related with the metal ion concentration, electronshell number and charge number of metal ion. Na+and Fe3+has little influence on theformation of nanotubes, but Cu2+, Mn2+, Ca2+, Zn2+with the electron shell number of3wasfeasible to form the nanotube. The addition of Ba2+induced the nano fiber formation ofovalbumin due to its electron shell number of5, and the addition of Mg2+had little influence onthe nanotube due to its electron shell number of2.
4) The formation mechanism of nanotubes in ovalbumin solution with addition of Cu2+,Mn2+, Ca2+, Zn2+under PEF treatment was proposed as followings: Firstly, the nano particlesformed with chelation of metal ions and protein under shorter PEF treatment time (1500s).Secondly, further treatment (2250s and3000s) induced the formation of ovalbuminnanofiber. Finally, the nanotubes formed by2or several nanofibers under PEF treatment forlonger time (3750s). The delicate nanotube also were observed with the size of20m length×200nm external diameter×30-10nm inner diameter. However, the nanotubes would beaggregated if too long PEF treatment were applied. Results verified that the present of metal ions was very important for forming nanotubes under PEF treatment. On the other hand, it wasfound that pH had little influence on the formation of protein nanotubes.
1，探索PEF对单一氨基酸的极化影响规律，结果如下：
（1）随着脉冲处理时间的延长（7.2ms到36ms），PEF诱使组氨酸、色氨酸、蛋氨酸分子中H-O-H伸缩振动，CH2反对称弯曲振动以及NH2剪刀弯曲振动呈现指数增强（Y=a ebx），而C-N伸缩振动和N-H弯曲振动出现指数（Y=a ln(bx)）减弱。同时，脉冲电场处理（电脉冲时间36ms）诱导组氨酸、蛋氨酸、色氨酸的1400cm-1附近峰消失，说明分子中C-O-O-H的弯曲和伸缩振动相对减弱。对应地，脉冲处理诱使3个单一氨基酸的HNMR图谱中所有H原子的分子缔合呈现指数（Y=a ebx）增强。同时，脉冲电场输入时间的增加诱导这3个单一氨基酸溶液电导率和pH值都略有增加。总结地，脉冲电场输入能量和单一氨基酸分子极化呈指数关系：Y=a ebx，即单一氨基酸分子以指数形式（Y=a ebx）接受电脉冲能量。
（2）对于组氨酸分子，当脉冲处理时间在7.2-28.8ms之间时，PEF对其的结构影响较小。然而较长时间（36ms）的PEF处理诱使组氨酸分子结构发生较大改变。推测36ms的PEF处理使得组氨酸聚集。
（3）对于蛋氨酸分子，FTIR和HNMR图谱均表明：PEF诱导蛋氨酸分子中S原子的极性发生变化，即脉冲电场引起S原子自旋耦合，S原子电子云发生变化。
（4）对于色氨酸分子，较短时间（7.2ms和14.4ms）的脉冲处理使得色氨酸分子活性增加，色氨酸吲哚环上的N基团极化，分子自旋耦合增强，即吲哚环中的N原子电子层发生变化。当较长时间（28.8ms和36ms）的脉冲处理使得色氨酸HNMR图谱裂分消失，色氨酸分子吲哚环上的电子云分布趋向稳定。
2，研究了脉冲电场对3肽，合成16肽，聚赖氨酸，-乳球蛋白的极化规律，结果如下：
（1）脉冲电场对氧化性谷胱甘肽分子结构的影响与脉冲电场对单一氨基酸的影响规律相似：氧化性谷胱甘肽分子结构变化和氢键缔合增强与电脉冲输入能量呈指数关系：(y=a ebx)。同时，脉冲电场对氧化性谷胱甘肽分子结构的影响和S原子关系密切。HNMR图谱也表明，脉冲电场诱导氧化性谷胱甘肽分子中S-S浓度呈对数减小，即PEF展开了氧化性谷胱甘肽分子中的二硫键。说明PEF诱导了S原子电子云分布发生变化，与PEF对蛋氨酸分子中S原子的影响相似。
（2）对于聚赖氨酸分子，较短时间（小于50s）脉冲电场诱导聚赖氨酸分子聚集，且分子聚集呈现线性，分子内的H-O-H伸缩振动，NH2剪刀弯曲振动和CH2反对称弯曲振动减小，C-N伸缩振动和N-H弯曲振动增加。然而较高脉冲能量的输入（脉冲时间从75s－175s）使得聚赖氨酸分子变化不明显。结果表明，脉冲电场引起聚赖氨酸分子聚集。
（3）对于合成16肽，较短电脉冲处理使得合成16肽分子打开，脉冲电场诱导合成16肽中H-O-H伸缩振动，NH2剪刀弯曲振动，C-N伸缩振动，N-H弯曲振动，以及CH2反对称伸缩振动呈现(y=ax2+bx+c)趋势，当脉冲电场处理时间为480s时，FTIR吸收强度都达到最小，即合成16肽分子展开程度最大。当电脉冲时间继续延长（600s），合成16肽分子重新聚集。同时，脉冲电场处理(120s)诱使合成16肽的HNMR图谱中1.18ppm位置的吸收峰消失，而在1.46ppm，1.82ppm和2.70ppm位置各出现一个新峰，说明合成16肽分子的展开，且分子中二硫键展开。结合PEF对谷胱甘肽分子结构中二硫键的影响，进一步验证了脉冲电场对蛋白质分子结构的展开与二硫键的展开有重要联系。
（4）对于-乳球蛋白，随着脉冲电场处理时间的延长，脉冲电场诱导-乳球蛋白分子中H-O-H伸缩振动，NH2剪刀弯曲振动和CH2反对称弯曲振动先减小而后增加(y=ax2+bx+c)，而C-N伸缩振动和N-H弯曲振动出现先增大而后减小的趋势(y=ax2+bx+c)，进一步验证了脉冲电场诱导-乳球蛋白分子先打开（小于100s）而后聚集（大于100s）。PEF对-乳球蛋白结构的影响和其对合成16肽的结构影响相似。
3，研究了脉冲电场对卵清蛋白分子结构的影响规律和脉冲电场制备卵清蛋白纳米管的机理探索，结果如下：
（1）脉冲电场输入能量和卵清蛋白的二级结构展开存在线性关系，卵清蛋白二级结构的展开是一个由三级结构到二级结构逐层逐步的过程。且在相同脉冲能量输入的情况下，蛋白质分子结构的展开对高电场强度的脉冲能量激发更敏感，而不是较长时间PEF处理的脉冲能量积累。相对-折叠结构易受热效应的影响，而-螺旋结构则更容易接受电脉冲作用，因为PEF对-螺旋结构中偶极距影响较大。
（2）脉冲电场和电导率对卵清蛋白二级结构的协同影响也呈线性关系。对于相同的PEF处理条件，电导率越高，即Ca2+浓度越高，蛋白质结构改变也越大。对于电导率为302S/cm的溶液接收脉冲电场处理3375s，和电导率为403S/cm的溶液接收脉冲电场处理3750s，以及电导率为516S/cm的溶液接收脉冲电场处理2250s和3000s，卵清蛋白溶液中都有蛋白质纳米管生成。特别地，当电导率为516μS/cm和606μS/cm的溶液接收3000s的PEF处理时，蛋白质纳米管形状清晰规范，纳米管长度在10m左右，外径在200nm左右，内径在30-50nm之间。当脉冲电场处理时间较长（4500s）和溶液电导率较高（816μS/cm）时，卵清蛋白分子聚集。结果说明，卵清蛋白纳米管的形成与脉冲电场参数和Ca2+浓度有关。
（3）对于添加不同摩尔比金属离子的卵清蛋白溶液，脉冲电场输入的脉冲能量和蛋白质分子的展开程度也成线性关系：[q]obs=a·Q+b。在脉冲电场处理条件下，蛋白质纳米管的形成不仅和金属离子的浓度有关，也和金属离子的带电荷数，金属离子的电子层数有关。一价金属离子（Na+）和三价金属离子（Fe3+）对蛋白质纳米管形成的作用不大。对于相同带电荷数的Mg2+，Zn2+，Ba2+，Mn2+，Cu2+和Ca2+而言，Cu2+，Mn2+，Ca2+，Zn2+更容易形成结构清晰的蛋白质纳米管，且它们都由3层电子层组成。Ba2+促使形成的是蛋白质纳米纤维，可能因为拥有5层电子层的原故。相对而言，Mg2+对蛋白质纳米管的形成影响不大，可能因为只有2层电子层。
（4）在脉冲电场处理条件下，对于添加Cu2+，Mn2+，Ca2+，Zn2+的卵清蛋白溶液，蛋白质纳米管的形成是一个递进过程。最初金属离子和蛋白质螯合形成纳米颗粒核，然后在纳米颗粒核的基础上，在脉冲电场的驱动下，蛋白质分子自组装成纳米线，继而2条或几条蛋白质纳米线自组装成蛋白质纳米管。所形成的蛋白质纳米管形状：精致规范，外径在200－300nm之间，内径在20－100nm之间，管长20m左右。当继续施加电脉冲能量时，蛋白质纳米管开始聚集，蛋白质分子变性。蛋白质分子聚集意味着蛋白质分子的变性，但蛋白质纳米管的形成并不意味着蛋白质分子变性，而是蛋白质分子结构改变后的最稳定分子状态，对应分子自由能最小。
（5）脉冲电场和pH协同作用不能形成蛋白质纳米管，然而PEF诱使pH12的卵清蛋白溶液中有蛋白质纳米纤维生成，再次证明蛋白质纳米管的形成需要金属离子作为桥梁。
The effect of pulsed electric fields (PEF) treatments on the molecular structurepolarization of histidine, methionine, tryptophan, Oxidative glutathione (GSSG), synthesized16-peptide and polylysine were investigated in this paper using Attenuated Total InternalReflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR), Proton Nuclear MagneticResonance Spectra (HNMR), Circular Dichroism (CD) and High Performance LiquidChromatography (HPLC). Accordingly, a new application of PEF as a novel technology on thepreparation of ovalbumin nanotubes was developed. The results were listed as followings:
1. Firstly, the effect of PEF treatment on the structure of single amino acid, such ashistidine, tryptophan and methionine, were investigated by FTIR and NMR. When the pulsedelectric time increased from7.2ms to36ms, the PEF treatment induced all stretching vibrationof H-O-H, anti-symmetric bending vibration of CH2and shear-bending vibration of NH2inFTIR spectra increased expontentially. However, the stretching vibration of C-N and bendingvibration of N-H decreased expontentially. When the longest PEF treatment time was applied(36ms), the peak at around1400cm-1disappreared which implied a significant decrease in thebending and stretching vibration of C-O-O-H. While in the HNMR spectra, it was observedthat all peaks shifted toward the lower wavenumber. It was also observed that PEF treatmentsresulted in a little increase in the conductivity and pH value of these three amino acids solution.
1) Especially as for the histidine, when the pulse time increased from7.2ms to28.8ms,the PEF treatment has little influence on its molecular structure. However, when the electricpulse treatment time increased to36ms, its influrence became significant. PEF treatment mayinduced a aggregation of histidine.2) As for the methionine, it was observed from both FTIRand HNMR spectra that PEF induced significant polarization of S atom.3) As for thetryptophan, when the electric pulse time increased to7.2ms and14.4ms, PEF induced thepolarization of N in the indole ring.
2. Thereafter, the effect of PEF on the oxidative glutathione (GSSG) was explored. FTIRanalysis demonstrated that an expontential relationship between the electric pulse energy and the change degree of molecular structure of oxidative glutathione (GSSG) was observed. WhileHNMR spectra implied that the S-S bonds in GSSG unfolded, which can be used to explain themechanism of the effect of PEF on Met.
3. The effect of PEF treatment on the structure of polylysine was inverstigated. Resutlsshowed that the shorter time (50s) PEF treatment induced the linear aggragation ofpolylysine. All peaks in the FTIR spetra about the stretching vibration of H-O-H, anti-symmetric bending vibration of CH2, the shear-bending vibration of NH2, the stretchingvibration of C-N and bending vibration of N-H presented an increase trend. However, when thetreatment time increased from75s to175s, the effect of PEF has little influence onpolylysine.
4. Following, the effect of PEF treatment on the structure of synthetic16peptide wasstudied. Results showed that the shorter PEF treatment time induced its unfolding as well.When the treatment time increased to480s, the most significant unfolding of16peptidestructure was observed. However, when the treatment time further increased to600s,apparently aggregation was observed. Similarly, HNMR spectra demonstrated the unfolding ofS-S bond among16peptide.
5. Furthermore,-lactoglobulin was chosen as the reoresentive to identify the effect ofPEF treatment on the structure of real protein. Results from FTIR analysis showed that with theincrease of PEF treatment time from25s to125s, the stretching vibration of H-O-H, anti-symmetric bending vibration of CH2, the shear-bending vibration of NH2, the stretchingvibration of C-N and bending vibration of N-H changed correspondingly.w When the electricpulse time continucely increased, the FTIR intensity of these above group decreased. Resultssuggested that PEF induced firstly unfolding and secondarily refolding of-lactoglobulin.
6. Finally, the effect of PEF on the molecular structure of ovalbumin and the mechanismof preparation ovalbumin nanotubes were studied.
1) Modulating the input energy via changing pulse intensity and treatment time, astepwise linear unfolding of secondary and tertiary structure of ovalbumin was observed. It isexplored that such change is more liable to electric pulse excitation rather than pulse energy accumulation, and the unfolding degree of tertiary structure was more significant than that ofsecondary structure. Compared with the-sheet was liable to the heat effective, the-helixwas more subjected to the electric pulse treatment due to its dipole moment structure.
2) A linear relationship between electric pulse energy and unfolding structure ofovalbumin were also observed. Under the same PEF treatment condition, the higherconductivity with higher Ca2+concentration, the more changes on the secondary structure ofovalbumin were obtained. It was found that when the sample was treated at the conductivity of302μS/cm or403μS/cm for3375s, or at516μS/cm for2250s, the formation ofovalbuminna notubes were observed. Especially for the PEF treatment with conductivity of516μS/cm or606μS/cm for3000s, the delicate nanotube were observed with the size of10m length×200nm external diameter×30-50nm inner diameter. However, under moresevere condition such as816μS/cm for4500s, the ovalbumin aggregated.
3) For the ovalbumin solution with difference metal ions concentration, the linearunfolding of secondary structure with increasing electric pulse energy was observed. At thesame time, the formation of nanotubes was related with the metal ion concentration, electronshell number and charge number of metal ion. Na+and Fe3+has little influence on theformation of nanotubes, but Cu2+, Mn2+, Ca2+, Zn2+with the electron shell number of3wasfeasible to form the nanotube. The addition of Ba2+induced the nano fiber formation ofovalbumin due to its electron shell number of5, and the addition of Mg2+had little influence onthe nanotube due to its electron shell number of2.
4) The formation mechanism of nanotubes in ovalbumin solution with addition of Cu2+,Mn2+, Ca2+, Zn2+under PEF treatment was proposed as followings: Firstly, the nano particlesformed with chelation of metal ions and protein under shorter PEF treatment time (1500s).Secondly, further treatment (2250s and3000s) induced the formation of ovalbuminnanofiber. Finally, the nanotubes formed by2or several nanofibers under PEF treatment forlonger time (3750s). The delicate nanotube also were observed with the size of20m length×200nm external diameter×30-10nm inner diameter. However, the nanotubes would beaggregated if too long PEF treatment were applied. Results verified that the present of metal ions was very important for forming nanotubes under PEF treatment. On the other hand, it wasfound that pH had little influence on the formation of protein nanotubes.
引文
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