Department of Chemistry, University of Nebraska, Lincoln, NE, United States.
Clin Chim Acta. 2011 Aug 17;412(17-18):1606-15. doi: 10.1016/j.cca.2011.05.012. Epub 2011 May 13.
The glycation of human serum albumin (HSA) during diabetes can affect the ability of this protein to bind drugs and small solutes in blood. This study describes the use of (16)O/(18)O-labeling and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to compare the levels of modification that occur throughout HSA under various glycation conditions in vitro. These quantitative studies build on a recent report that has identified the early and advanced glycation products that are formed on such samples of HSA.
Glycated HSA samples were prepared by incubating 42 g/l HSA with 0 to 15 mmol/l glucose at pH 7.4 and 37°C for up to 5 weeks. A control HSA sample was digested in (16)O-enriched water and glycated HSA samples were digested in the presence of (18)O-enriched water. These 2 types of samples were then mixed and the amounts of (16)O- vs. (18)O-labeled peptides were measured to determine the levels of modification that were occurring throughout HSA.
The largest levels of modification occurred in residues 101-119, 1-10 or 42-51, 87-100, 360-372, 521-531, and 275-286 of HSA after 2 weeks of glycation, and in residues 21-41, 1-10 or 42-51, 521-531, 82-93, and 146-160 after 5 weeks of glycation. Some of these regions contained the N-terminus, K199, K439, and K525, which have been previously identified as major glycation sites on HSA. The glycation pattern of HSA was dominated by early glycation products (e.g., fructosyl-lysine) after a reaction period of 2 weeks for mildly glycated HSA, while advanced glycation end products became more prominent at longer reaction times.
The time course of the observed modifications indicated that the pattern of glycation products changed as HSA was incubated over longer periods of time with glucose. Several regions found to have significant levels of modification were at or near the major drug binding regions on HSA. These results explain why the interaction of some drugs with HSA has been observed to vary with the level of glycation for this protein.
在糖尿病期间，人血清白蛋白（HSA）的糖化作用会影响该蛋白结合血液中药物和小分子的能力。本研究描述了使用（16）O/（18）O 标记和基质辅助激光解吸/电离飞行时间质谱，比较体外不同糖化条件下 HSA 中发生的修饰水平。这些定量研究建立在最近的一份报告的基础上，该报告确定了在这种 HSA 样本上形成的早期和晚期糖基化产物。
将 42 g/L HSA 与 0 至 15 mmol/L 葡萄糖在 pH 7.4 和 37°C 下孵育长达 5 周，制备糖化 HSA 样品。对照 HSA 样品在（16）O 富集水中消化，糖化 HSA 样品在（18）O 富集水中消化。然后将这 2 种样品混合，并测量（16）O-与（18）O-标记肽的量，以确定 HSA 中发生的修饰水平。
糖化 2 周后，HSA 中修饰水平最大的区域为 101-119、1-10 或 42-51、87-100、360-372、521-531 和 275-286，糖化 5 周后修饰水平最大的区域为 21-41、1-10 或 42-51、521-531、82-93 和 146-160。这些区域中的一些包含 N 末端、K199、K439 和 K525，它们以前被确定为 HSA 上的主要糖化位点。在温和糖化 HSA 的反应期为 2 周时，HSA 的糖化模式主要由早期糖化产物（如果糖基赖氨酸）主导，而随着反应时间的延长，晚期糖基化终产物变得更加明显。
观察到的修饰的时间过程表明，随着 HSA 与葡萄糖孵育时间的延长，糖化产物的模式发生了变化。在 HSA 中发现有显著修饰水平的几个区域位于或接近 HSA 上的主要药物结合区域。这些结果解释了为什么一些药物与 HSA 的相互作用会随着该蛋白的糖化程度而变化。
