Calorimetric Studies On The Interaction Between Protein And Surfactants And Enzyme-catalyzed Reaction

The interaction of Bovine Serum Albumin (BSA) with one anionic surfactant Sodium dodecyl sulfate (SDS) and seven cationic surfactants Hexadecyl trimethyl ammonium Bromide (CTAB), N-cetyl-hydroxyethyl-dimethyl ammonium bromide (CHDAB), N-cetyl-dihydroxyethyl-methyl ammonium bromide (CDHAB), iV-tetradecyl- hydroxyethyl-dimethyl ammonium bromide (THDAB), N-tetradecyl dihydroxyethyl-methyl ammonium bromide (TDHAB), N-dodecyl-hydroxyethyl -dimethyl ammonium bromide (DHDAB), N-dodecyl-dihydroxyethyl-methyl ammonium bromide (DDHAB), in the Tris-HCl buffer solutions (pH=7.0) has been investigated by isothermal titration calorimetry (ITC) at 298.15 to 318.15K. The binding site number, binding constant and thermodynamic function change were obtained by fitting the experimental data. The results show: there were two classes of binding sites on BSA molecules with SDS/CTAB/CHDAB/CDHAB/THDAB/ TDHAB, high affinity site and low affinity site. The high affinity binding were caused by electrostatic interaction of surfactant head groups with ionic sites on the protein molecules. The binging constant is larger on value and the binding process is characterized by exothermic enthalpy changes. The low affinity binding were due to the binding of alkyl chains of the surfactant to hydrophobic cavities of the protein molecules. The binding constant is smaller on value and the binding process causes positive thermal effect. But the binding BSA with DHDAB/DDHAB only has one high binding site.The Gibbs energy for the BSA binding to surfactants are all negative, but the entropy effects are positive. The binding constant decreases with the increase of the number of hydrophile groups in the surfactants, while becomes smaller with the increase of temperature, which would lead to increase of endothermic contribution and reduction of exothermic contribution and entropy. For the high affinity sites, binding constant decreases with the decrease of the alkyl chain lengh in the surfactants, but for the low affinity sites, binding constant increases and are not distinct when the alkyl chain is dedocyl. The binding sites are related to the alkyl chain lengh, not for the number of hydrophile groups and temperature.Isothermal titration calorimetry (ITC) has been used to observe the enzyme-catalyzed hydrolysis reaction of urea. Two kinetic parameters, the enzymatic conversion rate constant, k_cat, and the Maichaelis constant, K_M, for the urea (147 mmol·L^-1) / urease (3.0×10^-7 mmol·L^-1) enzymatic system in sodium phosphate buffer at pH 7.0 at T=(298.15 to 318.15)K are determined from the isothermal titration calorimetry experimental data. The results indicate that the urease-catalyzed reaction is well approximated by the Michaelis-Menten equation under the experimental conditions. The effect of the temperature on the variation of the values of K_cat can be correlated quantitatively to the Arrhenius equation with the apparent activation energy of 16.6 kJ·mol^-1. The results show a good example of effectively evaluating kinetic parameters for enzyme-catalyzed reaction by ITC. It is suggested that ITC should be suitable and have significant potential for the kinetic study of enzyme activity.Isothermal titration calorimetry (ITC) has been used to observe the human cytochrome CYP3A4 (3.6×10^-7mmol·L^-1) catalyzed reaction of aminobenzimidazole and 1-methyl-aminobenzimidazole (5 mmol·L^-1) at 310.15K (pH=7.4 PB buffer). The results indicate that the exothermic contribution of CYP3A4 catalyzed is too small cunder the experimental conditions, metabolizing reaction is not distinct, The exothermic contribution of 1-methyl-aminobenzimidazole catalyzed by CYP3A4 is large than aminobenzimidazole, binding interaction (nonspecific binding) is likely to occur and we should do the research profundity.