Duff Michael R, Kumar Challa V
Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269-3060, USA.
Langmuir. 2009 Nov 3;25(21):12635-43. doi: 10.1021/la901901k.
The interaction of proteins with a solid surface involves a complex set of interactions, and elucidating the details of these interactions is essential in the rational design of solid surfaces for applications in biosensors, biocatalysis, and biomedical applications. We examined the enthalpy changes accompanying the binding of met-hemoglobin, met-myoglobin, and lysozyme to layered alpha-Zr(IV)phosphate (20 mM NaPipes, 1 mM TBA, pH 7.2, 298 K) by titration calorimetry, under specific conditions. The corresponding binding enthalpies for the three proteins are -24.2 +/- 2.2, -10.6 +/- 2, and 6.2 +/- 0.2 kcal/mol, respectively. The binding enthalpy depended on the charge of the protein where the binding of positively charged proteins to the negatively charged solid surface was endothermic while the binding of negatively charged proteins to the negatively charged solid was exothermic. These observations are contrary to a simple electrostatic model where binding to the oppositely charged surface is expected to be exothermic. The binding enthalpy depended on the net charge on the protein, ionic strength of the medium, the type of buffer ions present, and temperature. The temperature dependence studies of binding enthalpies resulted in the estimation of heat capacity changes accompanying the binding. The heat capacity changes observed with Hb, Mb, and lysozyme are 1.4 +/- 0.3, 0.89 +/- 0.2, and 0.74 +/- 0.1 kcal/(mol.K), respectively, and these values depended on the net charge of the protein. The enthalpy changes also depended linearly on the enthalpy of ionization of the buffer, and the numbers of protons released per protein estimated from this data are 12.6 +/- 2, 6.0 +/- 1.2, and 1.2 +/- 0.5 for Hb, Mb, and lysozyme, respectively. Binding enthalpies, independent of buffer ionization, are also estimated from these data. Entropy changes are related to the loss in the degrees of freedom when the protein binds to the solid and the displacement of solvent molecules/protons/ions from the protein-solid interface. Proton coupled protein binding is one of the major processes in these systems, which is novel, and the binding enthalpies can be predicted from the net charge of the protein, enthalpy of buffer ionization, ionic strength, and temperature.
蛋白质与固体表面的相互作用涉及一系列复杂的相互作用，阐明这些相互作用的细节对于合理设计用于生物传感器、生物催化和生物医学应用的固体表面至关重要。我们通过滴定热分析法，在特定条件下研究了高铁血红蛋白、高铁肌红蛋白和溶菌酶与层状α-磷酸锆（20 mM 哌嗪-N,N'-双（2-乙磺酸），1 mM 四丁基铵，pH 7.2，298 K）结合时的焓变。三种蛋白质相应的结合焓分别为-24.2±2.2、-10.6±2和6.2±0.2 kcal/mol。结合焓取决于蛋白质的电荷，其中带正电荷的蛋白质与带负电荷的固体表面结合是吸热的，而带负电荷的蛋白质与带负电荷的固体结合是放热的。这些观察结果与简单的静电模型相反，在简单静电模型中，与带相反电荷的表面结合预计是放热的。结合焓取决于蛋白质上的净电荷、介质的离子强度、存在的缓冲离子类型以及温度。结合焓的温度依赖性研究得出了结合时伴随的热容变化估计值。高铁血红蛋白、肌红蛋白和溶菌酶观察到的热容变化分别为1.4±0.3、0.89±0.2和0.74±0.1 kcal/(mol·K)，这些值取决于蛋白质的净电荷。焓变还与缓冲液的电离焓呈线性关系，根据该数据估算出每种蛋白质释放的质子数分别为高铁血红蛋白12.6±2、肌红蛋白6.0±1.2和溶菌酶1.2±0.5。还从这些数据中估算了与缓冲液电离无关的结合焓。熵变与蛋白质结合到固体时自由度的损失以及溶剂分子/质子/离子从蛋白质-固体界面的位移有关。质子偶联蛋白质结合是这些系统中的主要过程之一，这是新颖的，并且结合焓可以根据蛋白质的净电荷、缓冲液电离焓、离子强度和温度来预测。
