Department of Physiological Chemistry, Osaka Medical College, 2-7 Daigakucho, Takatsuki 565-8686, Japan.
Clin Chim Acta. 2010 Oct 9;411(19-20):1536-8. doi: 10.1016/j.cca.2010.05.044. Epub 2010 Jun 4.
In clinical settings, human serum albumin (HSA) is widely quantitated on the basis of change in the color of bromocresol purple (BCP) bound to HSA. However, the binding sites of BCP on HSA are hitherto unknown. We have identified these sites by performing binding experiments in which BCP was bound to HSA complexed with warfarin, ibuprofen, propofol, or myristic acid.
The binding experiment was performed with HSA complexed with a drug in buffers at pH 4.8 and 7.2. The number of BCP binding sites (n) and the association constant (Ka) were obtained by Scatchard analysis using the absorbance data of the BCP-HSA complexes.
Two BCP binding sites on native HSA were identified at pH 7.2 and 1.3 sites were identified at pH 4.8. The number of sites for warfarin-HSA binding was approximately half that of native HSA at both pH values. The site numbers for myristate- and ibuprofen-HSA were 0.3 and 0.8 at pH 7.2, and 1.5 and 2.0 at pH 4.8, respectively; their Ka values were also high (18 x 10(4) and 10 x 10(4)). The Ka values for all HSA samples, except for the 2 samples, arranged from 1.5 x 10(4) to 3.3 x 10(4). The n and Ka values for propofol were equivalent to those for native HSA.
We postulate that the 2 BCP binding sites include a warfarin-binding site (drug-binding site 1) and 1 of the 2 ibuprofen-binding sites.
在临床环境中，人血清白蛋白（HSA）的浓度通常基于与 HSA 结合的溴甲酚绿（BCP）颜色变化来定量。然而，BCP 与 HSA 的结合位点至今仍不清楚。我们通过进行结合实验来确定这些结合位点，在该实验中，BCP 与华法林、布洛芬、丙泊酚或豆蔻酸与 HSA 形成的复合物结合。
在 pH 值为 4.8 和 7.2 的缓冲液中，将 HSA 与药物复合物进行结合实验。通过使用 BCP-HSA 复合物的吸光度数据，使用 Scatchard 分析获得 BCP 的结合位点数（n）和结合常数（Ka）。
在 pH 值为 7.2 时，鉴定出天然 HSA 上有两个 BCP 结合位点，在 pH 值为 4.8 时鉴定出 1.3 个结合位点。在这两种 pH 值下，华法林-HSA 结合的结合位点数约为天然 HSA 的一半。豆蔻酸-HSA 和布洛芬-HSA 的结合位点数在 pH 值为 7.2 时分别为 0.3 和 0.8，在 pH 值为 4.8 时分别为 1.5 和 2.0；它们的 Ka 值也很高（18×104 和 10×104）。除了 2 个样本外，所有 HSA 样本的 Ka 值在 1.5×104 到 3.3×104 之间。丙泊酚的 n 和 Ka 值与天然 HSA 相当。
我们假设这两个 BCP 结合位点包括一个华法林结合位点（药物结合位点 1）和两个布洛芬结合位点之一。
