Flora K, Brennan J D, Baker G A, Doody M A, Bright F V
Department of Chemistry, Brock University, St. Catharines, Ontario L2S 3A1, Canada.
Biophys J. 1998 Aug;75(2):1084-96. doi: 10.1016/S0006-3495(98)77598-8.
Steady-state and time-resolved fluorescence spectroscopy was used to follow the local and global changes in structure and dynamics during chemical and thermal denaturation of unlabeled human serum albumin (HSA) and HSA with an acrylodan moiety bound to Cys34. Acrylodan fluorescence was monitored to obtain information about unfolding processes in domain I, and the emission of the Trp residue at position 214 was used to examine domain II. In addition, Trp-to-acrylodan resonance energy transfer was examined to probe interdomain spatial relationships during unfolding. Increasing the temperature to less than 50 degrees C or adding less than 1.0 M GdHCl resulted in an initial, reversible separation of domains I and II. Denaturation by heating to 70 degrees C or by adding 2.0 M GdHCl resulted in irreversible unfolding of domain II. Further denaturation of HSA by either method resulted in irreversible unfolding of domain I. These results clearly demonstrate that HSA unfolds by a pathway involving at least three distinct steps. The low detection limits and high information content of dual probe fluorescence should allow this technique to be used to study the unfolding behavior of entrapped or immobilized HSA.
稳态和时间分辨荧光光谱法被用于追踪未标记的人血清白蛋白（HSA）以及与丙烯基丹部分结合于半胱氨酸34位点的HSA在化学和热变性过程中结构与动力学的局部和整体变化。监测丙烯基丹荧光以获取有关结构域I展开过程的信息，并用214位色氨酸残基的发射来检测结构域II。此外，还检测了色氨酸到丙烯基丹的共振能量转移，以探测展开过程中的结构域间空间关系。将温度升高至低于50℃或添加低于1.0 M的GdHCl会导致结构域I和II最初的、可逆的分离。加热至70℃或添加2.0 M的GdHCl进行变性会导致结构域II不可逆的展开。通过这两种方法对HSA进一步变性会导致结构域I不可逆的展开。这些结果清楚地表明，HSA通过一个至少涉及三个不同步骤的途径展开。双探针荧光的低检测限和高信息含量应使该技术可用于研究被包封或固定化的HSA的展开行为。
