Cheema Mohammad Arif, Taboada Pablo, Barbosa Silvia, Castro Emilio, Siddiq Mohammad, Mosquera Víctor
Grupo de Física de Coloides y Polímeros, Departamento de Física de la Materia Condensada, Facultad de Física, Universidad de Santiago de Compostela, E-15782, Santiago de Compostela, Spain.
Biomacromolecules. 2007 Aug;8(8):2576-85. doi: 10.1021/bm070354j. Epub 2007 Jun 26.
The interactions and complexation process of the amphiphilic phenothiazine fluphenazine hydrochloride with human serum albumin in aqueous buffered solutions of pH 3.0 and 7.4 have been examined by zeta-potential, isothermal titration calorimetry (ITC), UV-vis spectroscopy, and dynamic light scattering (DLS) techniques with the aim of analyzing the effect of hydrophobic and electrostatic forces on the complexation process and the alteration of protein conformation upon binding. Thus, the energetics and stoichiometry of the binding process were derived from ITC data. The enthalpies of binding obtained are small and exothermic, so the Gibbs energies of binding are dominated by large increases in entropy, consistent with hydrophobic interactions at a acidic pH. However, at physiological pH, binding to the first class of binding sites is dominated by an enthalpic contribution due to the existence of electrostatic interactions and probably some hydrogen bonding. Binding isotherms were obtained from microcalorimetric data by using a theoretical model based on the Langmuir isotherm. zeta-Potential data showed a reversal in the sign of the protein charge at pH 7.4, as a consequence of the binding of the drug to the protein. Gibbs energies of drug binding per mole of drug were also derived from zeta-potential data. On the other hand, binding of the phenothiazine that causes a conformational transition on the protein structure was followed as a function of drug concentration using UV-vis spectroscopy, and the data were analyzed to obtain the Gibbs energy of the transition in water (deltaG(degree)w) and in a hydrophobic environment (deltaG(degree)hc). Finally, the population distribution of the different species in solution and the size of the complexes were analyzed through dynamic light scattering. The existence of an aggregation process of drug/protein complexes, as a consequence of the expanded structure of the protein induced by the drug and subsequent further binding, is in agreement with ITC data. In addition, detection of drug aggregates at concentrations below the drug critical micelle concentration was also detected by this technique.
在pH为3.0和7.4的水性缓冲溶液中，通过zeta电位、等温滴定量热法（ITC）、紫外可见光谱和动态光散射（DLS）技术研究了两亲性吩噻嗪盐酸氟奋乃静与人血清白蛋白的相互作用和络合过程，目的是分析疏水和静电力对络合过程的影响以及结合后蛋白质构象的变化。因此，结合过程的能量学和化学计量学是从ITC数据中推导出来的。获得的结合焓很小且为放热，因此结合的吉布斯自由能主要由熵的大幅增加主导，这与酸性pH下的疏水相互作用一致。然而，在生理pH下，由于静电相互作用以及可能存在的一些氢键，与第一类结合位点的结合主要由焓贡献主导。通过使用基于朗缪尔等温线的理论模型，从微量热数据中获得结合等温线。zeta电位数据显示，由于药物与蛋白质的结合，在pH 7.4时蛋白质电荷的符号发生了反转。每摩尔药物的药物结合吉布斯自由能也从zeta电位数据中推导出来。另一方面，使用紫外可见光谱跟踪导致蛋白质结构发生构象转变的吩噻嗪的结合随药物浓度的变化，并对数据进行分析以获得在水中（ΔG°w）和疏水环境中（ΔG°hc）转变的吉布斯自由能。最后，通过动态光散射分析溶液中不同物种的群体分布以及复合物的大小。药物/蛋白质复合物聚集过程的存在，是由于药物诱导蛋白质结构扩展以及随后的进一步结合，这与ITC数据一致。此外，该技术还检测到在低于药物临界胶束浓度的浓度下存在药物聚集体。
