Chemistry Department, University of Nebraska, Lincoln, Lincoln, NE 68588-0304, USA.
J Pharm Biomed Anal. 2010 Nov 2;53(3):811-8. doi: 10.1016/j.jpba.2010.04.035. Epub 2010 May 6.
Diabetes leads to elevated levels of glucose in blood which, in turn, can lead to the non-enzymatic glycation of serum proteins such as human serum albumin (HSA). It has been suggested that this increase in glycation can alter the ability of HSA to bind to drugs and other small solutes. This study used high-performance affinity chromatography (HPAC) to see if there is any significant change related to glycation in the binding of HSA to warfarin and l-tryptophan, which are often used as probe compounds for Sudlow sites I and II of HSA in drug binding studies with this protein. It was found through frontal analysis studies that both of these compounds gave a good fit to a single-site binding model with glycated HSA under the conditions used in this study. There was no significant change in the association equilibrium constants or specific activities for warfarin with HSA at pH 7.4 and 37 degrees C under glycation conditions that were representative of those expected in pre-diabetes or diabetes, but a 4.7- to 5.8-fold increase in binding affinity for l-tryptophan with glycated HSA was observed. These results indicate that warfarin and l-tryptophan can be successively used as site-selective probes for glycated HSA; however, changes in the affinity of l-tryptophan may need to be considered in such an application. These results should be valuable in future competition studies using these compounds as probes to examine the interactions of other drugs and solutes with Sudlow sites I and II and to determine how changes in HSA glycation can affect the serum protein binding of various pharmaceutical agents during diabetes.
糖尿病导致血液中葡萄糖水平升高，这反过来又会导致血清蛋白（如人血清白蛋白（HSA））的非酶糖基化。有人认为，这种糖化程度的增加会改变 HSA 与药物和其他小分子溶质结合的能力。本研究使用高效亲和色谱（HPAC）来观察 HSA 与华法林和 l-色氨酸的结合是否存在与糖化相关的任何显著变化，华法林和 l-色氨酸通常被用作 HSA 与该蛋白结合研究中 Sudlow 部位 I 和 II 的探针化合物。通过前沿分析研究发现，在本研究中使用的条件下，这两种化合物都与糖化 HSA 很好地符合单点结合模型。在糖化条件下，Warfarin 与 HSA 的缔合平衡常数或比活性在 pH 7.4 和 37°C 下没有显著变化，这些条件代表了糖尿病前期或糖尿病中预期的条件，但观察到与糖化 HSA 的结合亲和力增加了 4.7 至 5.8 倍。这些结果表明，华法林和 l-色氨酸可以成功地连续用作糖化 HSA 的位点选择性探针；然而，在这种应用中，可能需要考虑 l-色氨酸亲和力的变化。这些结果对于未来使用这些化合物作为探针进行竞争研究以检查其他药物和溶质与 Sudlow 部位 I 和 II 的相互作用以及确定 HSA 糖化变化如何影响各种药物在糖尿病期间与血清蛋白结合的能力将非常有价值。
