Department of Chemistry, University of Nebraska, Lincoln, NE, 68588-0304, USA.
Department of Chemistry, University of Nebraska, Lincoln, NE, 68588-0304, USA.
J Chromatogr A. 2021 Feb 8;1638:461683. doi: 10.1016/j.chroma.2020.461683. Epub 2020 Nov 10.
Diabetes is characterized by elevated levels of blood glucose, which can result in the modification of serum proteins. The modification of a protein by glucose, or glycation, can also lead to the formation of advanced glycated end-products (AGEs). One protein that can be modified through glycation and AGE formation is human serum albumin (HSA). In this study, immunoextraction based on polyclonal anti-HSA antibodies was used with high-performance affinity microcolumns to see how AGE-related modifications produced by glyoxal (Go) and methylglyoxal (MGo) affected the binding of HSA to several first- and second-generation sulfonylureas, a class of drugs used to treat type II diabetes and known to bind to HSA. With this approach, it was possible to use a single platform to examine drug interactions with several preparations of HSA. Each applied protein sample could be used over 20-50 experiments, and global affinity constants for most of the examined drugs could be obtained in less than 7.5 min. The binding constants measured for these drugs with normal HSA gave good agreement with global affinities based on the literature. Both Go- and MGo-related modifications at clinically relevant levels were found by this method to create significant changes in the binding by some sulfonylureas with HSA. The global affinities for many of the drugs increased by 1.4-fold or more; gliclazide and tolazamide had no significant change with some preparations of modified HSA, and a small-to-moderate decrease in binding strength was noted for glibenclamide and gliclazide with Go-modified HSA. This approach can be adapted for the study of other drug-protein interactions and alternative modified proteins by altering the antibodies that are employed for immunoextraction and within the affinity microcolumn.
糖尿病的特征是血液葡萄糖水平升高，这可能导致血清蛋白的修饰。葡萄糖或糖基化对蛋白质的修饰也会导致晚期糖基化终产物 (AGE) 的形成。一种可以通过糖基化和 AGE 形成修饰的蛋白质是人血清白蛋白 (HSA)。在这项研究中，使用基于多克隆抗 HSA 抗体的免疫提取法和高性能亲和微柱来观察乙二醛 (Go) 和甲基乙二醛 (MGo) 产生的与 AGE 相关的修饰如何影响 HSA 与几种第一代和第二代磺酰脲类药物的结合，磺酰脲类药物是一类用于治疗 2 型糖尿病的药物，已知与 HSA 结合。通过这种方法，可以使用单个平台来检查几种 HSA 制剂与药物的相互作用。每个应用的蛋白质样品可以在 20-50 次实验中使用，并且可以在不到 7.5 分钟的时间内获得大多数检查药物的全局亲和力常数。用正常 HSA 测量的这些药物的结合常数与文献中基于全局亲和力的常数很好地吻合。在临床相关水平上，通过这种方法发现 Go 和 MGo 相关修饰会导致一些磺酰脲类药物与 HSA 结合发生显著变化。许多药物的全局亲和力增加了 1.4 倍或更多；对于某些修饰的 HSA，格列齐特和甲苯磺丁脲的结合没有明显变化，而对于 Go 修饰的 HSA，格列本脲和格列齐特的结合强度略有下降。通过改变用于免疫提取的抗体和亲和微柱内的抗体，这种方法可以适用于其他药物-蛋白质相互作用和替代修饰蛋白质的研究。
