Salt‐dependent monomer–dimer equilibrium of bovine β‐lactoglobulin at pH 3
Salt‐dependent monomer–dimer equilibrium of bovine β‐lactoglobulin at pH 3
复制标题
pH 3 时牛 β-乳球蛋白的盐依赖性单体-二聚体平衡
作者：
K. Sakurai;M. Oobatake;Y. Goto
虽然牛β-乳球蛋白在无盐的情况下，在pH值为3时呈现单体天然结构，但盐的添加稳定了二聚体。用沉降法研究了盐浓度对单体-二聚体平衡的影响。在1M以下加入氯化钠、氯化钾或盐酸胍，以类似的方式稳定二聚体。另一方面，NaClO4的效果比其他盐高约20倍，这表明阴离子结合是盐诱导二聚体形成的原因，就像在酸未折叠蛋白质中观察到的那样。在5M加入盐酸胍，由于蛋白质结构的变性，使二聚体解离成单体。在NaC l或NaClO_4存在下，二聚反应常数随温度的升高而减小，说明二聚体的生成热变(Δ-HD)为负值。在pH值为3.0时用NaClO4滴定单体β-乳球蛋白，用等温滴定量热仪直接测量二聚体形成的热效应。减去盐稀释热后的净热效应(对应于ΔHD)为负，与沉积平衡得到的结果一致。根据沉淀平衡测得的二聚常数与温度的关系，估算了20℃时的ΔHd值和二聚体的生成热容变化(ΔCp)。在NaC l和NaClO_4中，测得的ΔCp均为负值，表明疏水表面的埋藏对二聚体的形成起主导作用。观察到的ΔCp值与用可接近表面积法计算的X射线二聚体结构的值一致。这些结果表明，在pH值为3时，β-乳球蛋白的单体-二聚体平衡是由疏水和静电效应的微妙平衡决定的，而疏水和静电效应受盐的添加或温度变化的影响。
Although bovine β‐lactoglobulin assumes a monomeric native structure at pH 3 in the absence of salt, the addition of salts stabilizes the dimer. Thermodynamics of the monomer–dimer equilibrium dependent on the salt concentration were studied by sedimentation equilibrium. The addition of NaCl, KCl, or guanidine hydrochloride below 1 M stabilized the dimer in a similar manner. On the other hand, NaClO4 was more effective than other salts by about 20‐fold, suggesting that anion binding is responsible for the salt‐induced dimer formation, as observed for acid‐unfolded proteins. The addition of guanidine hydrochloride at 5 M dissociated the dimer into monomers because of the denaturation of protein structure. In the presence of either NaCl or NaClO4, the dimerization constant decreased with an increase in temperature, indicating that the enthalpy change (ΔHD) of dimer formation is negative. The heat effect of the dimer formation was directly measured with an isothermal titration calorimeter by titrating the monomeric β‐lactoglobulin at pH 3.0 with NaClO4. The net heat effects after subtraction of the heat of salt dilution, corresponding to ΔHD, were negative, and were consistent with those obtained by the sedimentation equilibrium. From the dependence of dimerization constant on temperature measured by sedimentation equilibrium, we estimated the ΔHD value at 20°C and the heat capacity change (ΔCp) of dimer formation. In both NaCl and NaClO4, the obtained ΔCp value was negative, indicating the dominant role of burial of the hydrophobic surfaces upon dimer formation. The observed ΔCp values were consistent with the calculated value from the X‐ray dimeric structure using a method of accessible surface area. These results indicated that monomer–dimer equilibrium of β‐lactoglobulin at pH 3 is determined by a subtle balance of hydrophobic and electrostatic effects, which are modulated by the addition of salts or by changes in temperature.
固体模型化合物和蛋白质展开的热力学。
影响因子：
5.6
作者：
Murphy,KP;Gill,SJ
通讯作者：
Gill,SJ