The Effect Of Pei On Conformation And Activity Of Horseradish Peroxidase And Lactic Dehydrogenase

In recent years, polyethyleneimine(PEI) gene carrier material is attracting more and more interests due to the favourable transfection efficiency in the genetic engineering field. However, the theoretical evidence for the potential security problem is still unclear, especially the consensus among scientists and researchers respect to the effect mechanism of PEI on functional proteins (such as enzymes) is a blank hitherto. The spectrum of UV-vis absorption, fluorescence, and circular dichroism, the method of enzymatic reaction kinetics, and the ITC were used to research the effect of PEI on the molecular conformation, catalytic activity, thermalstability and the enzymatic reaction kinetics of horseradish peroxidase (HRP) and lactate dehydrogenase (LDH). Additionally, the interaction mechanisms between PEI and the two kinds of proteins were investigated. The theoretical evidence for the two enzymes is provided for the security problem of PEI as a gene carrier material in this paper. The original resualts from this research can be mainly concluded as follows:It is indicated that molecular structure and the biologic function of HRP was changed significantly by PEI. (1) The tertiary structure of protein molecule was affected remarkablely while the secondary structure affected weakly in the presence of PEI. The explanation for comformational changes could be the formation of the PEI-HRP complexes and the change of HRP peptide chain skeleton by PEI. Moreover, the hydrophobicity in vicinity near the tryptophan and tyrosine residues was changed and the degree of exposure in the polar environment for the two kinds of amino acids varied accordingly in the presence of PEI. (2) In simulated physiological solution, catalytic activity of HRP was enhanced by PEI, and the enzyme catalysis reaction kinetics parameters of Km and Vmax could be changed as well. While, PEI showed a negative effect for the thermostability of HRP. (3) The ITC experiment revealed that the interaction between PEI and HRP showed a significant thermal effect. In the first step, it was shown that ΔH1>0, ΔS1>0, and in the second step ΔH2<0, ΔS2<0. It was indicated that the interaction was mainly contributed by hydrophobic interaction force, hydrogen bond and Van der Waals force.The conformation and catalytic function of LDH was also clearly changed in the presence of PEI in simulated physiological solution. (1) The catalytic activity of LDH was decreased under the condition of determination in 30℃, and the catalytic reaction kinetic parameters of Km and Vmax varied as well in the presence of the PEI. Meanwhile, the heat-resistant stability of LDH was weakened obviously. (2) PEI showed a remarkable impact on both the secondary and tertiary structure of LDH, and the microenvironmental changes near the tryptophan and tyrosine residues of LDH were showed significantly. In addition, the degree of exposure in the polar solvent for the two kinds of amino acid groups decreased. It is likely that the two kinds of moleculars could be formed the ground state complexs. (3) According to the ITC experiment, the interaction between PEI and LDH was an endothermic process. Meanwhile, it was an entropically driven process because the interaction Thermodynamic parameters showed that:ΔH>0, AS>0. It was indicated that the hydrophobic interaction foce played a major role in the reaction.