Isothermal titration calorimetric studies of the pH induced conformational changes of bovine serum albumin
Isothermal titration calorimetric studies of the pH induced conformational changes of bovine serum albumin
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DOI：
10.1007/s10973-009-0040-5
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发表时间：
2009-06-01
影响因子：
4.4
通讯作者：
Dekany, Imre
中科院分区：
文献类型：
--
作者：
Kun, Robert;Szekeres, Marta;Dekany, Imre
牛血清白蛋白 (BSA) 是一种软球状蛋白质，在 pH 范围 2-13.5 内通过几个确定的转变步骤发生构象变化。改变构象的能力使 BSA 能够复合不同的配体，从脂肪酸到阳离子或药物，并将它们携带到血液中。用 NaOH 溶液微量热滴定 BSA，以测量构象变化的焓。在pH 3和9.5之间的滴定过程中发现了两次放热焓变，这可以通过E-F和F-N转变来识别。 pH 3.5 时的焓变（BSA 从 E 形式转变为 F 形式，结构域 I 中结构域内螺旋的折叠）与蛋白质浓度无关。对于 0.1% BSA 溶液，在 pH 4.8 时观察到第二个转变（F-N，结构域 III 的折叠），但随着蛋白质浓度增加到 0.2% 和 0.3%，它转变为更高的 pH 值。通过测量色氨酸残基的内在荧光，验证了蛋白质结构随着 pH 值的增加而收紧。在更高的 pH 值（pH 10.5）下，荧光测量显示蛋白质膨胀。还通过动态光散射测量了 BSA 构象变化。 i.e.p 处的流体动力学直径较小。 BSA（pH 值接近 5 时为 5-7 nm），并且在 pH 范围的两端更大（pH 2 时为 17.5 nm，pH 10 时为 8.3 nm）。
Bovine serum albumin (BSA) is a soft globular protein that undergoes conformational changes through several identified transition steps in the pH range 2-13.5. The ability to change conformation makes BSA capable of complexing different ligands from fatty acids to cations or drugs and carries them in the bloodstream. Microcalorimetric titration of BSA with NaOH solution was performed to measure the enthalpy of conformational changes. Two exothermic enthalpy changes were found in the course of the titration between pH 3 and 9.5, which can be identified with the E-F, and the F-N transitions. The enthalpy change at pH 3.5 (transition from the E to the F form of BSA, folding of intra-domain helices in domain I) is independent of the protein concentration. The second transition (F-N, folding of domain III) was observed at pH 4.8 for the 0.1% BSA solution, but it shifted to higher pH values as the protein concentration increased to 0.2% and 0.3%. The tightening of the protein structure with increasing pH was verified measuring intrinsic fluorescence of tryptophan residues. At even higher pH value, pH 10.5, fluorescence measurements revealed protein expansion. The BSA conformational changes were also measured by dynamic light scattering. The hydrodynamic diameter was smaller at the i.e.p. of BSA (5-7 nm at pH similar to 5) and larger at the two ends of the pH range (17.5 nm at pH 2 and 8.3 nm at pH 10).