Effect Of Pulsed Electric Field On The Polarization Of Amino Acids And Preparation Of Protein Nanotube

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.