Department of Chemistry, University of Nebraska, Lincoln, NE 68588, USA.
Department of Chemistry, University of Nebraska, Lincoln, NE 68588, USA.
J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Jul 15;1226:123798. doi: 10.1016/j.jchromb.2023.123798. Epub 2023 Jun 10.
Modification of proteins can occur during diabetes due to the formation of advanced glycation end-products (AGEs) with reactive dicarbonyls such as glyoxal (Go) and methylglyoxal (MGo). Human serum albumin (HSA) is a serum protein that binds to many drugs in blood and that is known to be modified by Go and MGo. This study examined the binding of various sulfonylurea drugs with these modified forms of HSA by using high-performance affinity microcolumns prepared by non-covalent protein entrapment. Zonal elution experiments were employed to compare the retention and overall binding constants for the drugs with Go- or MGo-modified HSA vs normal HSA. The results were compared to values from the literature, such as measured or estimated using affinity columns containing covalently immobilized HSA or biospecifically-adsorbed HSA. The entrapment-based approach provided estimates of global affinity constants within 3-5 min for most of the tested drugs and with typical precisions of ±10-23%. Each entrapped protein microcolumn was stable for over at least 60-70 injections and one month of use. The results obtained with normal HSA agreed at the 95% confidence level with global affinity constants that have been reported for the given drugs in the literature. It was found for HSA that had been modified with clinically-relevant levels of either Go or MGo that an increase in the global affinity constant of up to 2.1-fold occurred for some of the tested drugs. The information acquired in this study can be used in the future to adapt this entrapment-based approach to study and evaluate interactions between other types of drugs and normal or modified binding agents for clinical testing and biomedical research.
蛋白质在糖尿病期间可能会发生修饰，这是由于形成了具有反应性二羰基化合物（如乙二醛（Go）和甲基乙二醛（MGo））的高级糖基化终产物（AGEs）。人血清白蛋白（HSA）是一种与血液中许多药物结合的血清蛋白，已知其可被 Go 和 MGo 修饰。本研究通过使用非共价蛋白包埋制备的高性能亲和微柱，研究了各种磺酰脲类药物与这些修饰形式的 HSA 的结合。采用区带洗脱实验比较了药物与 Go 或 MGo 修饰的 HSA 与正常 HSA 的保留和整体结合常数。将结果与文献中的值进行比较，例如使用含有共价固定化 HSA 或生物特异性吸附 HSA 的亲和柱测量或估计的值。基于包埋的方法在大多数测试药物中，在 3-5 分钟内提供了全局亲和力常数的估计值，典型精度为±10-23%。每个包埋的蛋白质微柱在至少 60-70 次注射和一个月的使用中都保持稳定。用正常 HSA 获得的结果在 95%置信水平上与文献中报道的给定药物的全局亲和力常数一致。研究发现，对于已经用临床相关水平的 Go 或 MGo 修饰的 HSA，一些测试药物的全局亲和力常数增加了 2.1 倍。本研究获得的信息可用于将来适应这种基于包埋的方法，以研究和评估其他类型的药物与正常或修饰的结合剂之间的相互作用，用于临床测试和生物医学研究。
