Department of Pharmaceutical Sciences, North Dakota State University, Fargo, ND 58108, USA.
Clin Chim Acta. 2011 Nov 20;412(23-24):2105-16. doi: 10.1016/j.cca.2011.07.022. Epub 2011 Jul 29.
Serum protein glycation and formation of advanced glycation endproducts (AGE) correlates with diabetic complications. Highly AGE-modified albumin is frequently used to study the biochemical and cellular activities of AGE-proteins. However, moderately modified albumin samples are expected to be (patho)physiologically more relevant for diabetes research. We produced a panel of nine moderately modified albumin samples and characterized these in terms of side chain modifications, secondary structure, folding stability, and spectroscopic properties.
A panel of nine albumin samples modified with glucose, methylglyoxal, glyoxylic acid and carboxymethyl lysine was characterized in terms of side chain modifications, thermal folding stability, secondary structure, aggregation, surface charge, and ligand binding. The analytical tools employed included chemical analysis, biochemical and immunochemical assays for side chain modifications, near UV circular dichroism, differential scanning calorimetry, analytical size exclusion and ion exchange HPLC, and a ligand binding assay.
Moderate glycation and AGE modification of serum albumin causes structural changes that depend on the chemical reactivity of the modifying reagent and the concentration used for in-vitro glycation. In general, the α-helical content is decreased and thermal unfolding behavior is altered. However, moderate glycation does not cause aggregation or formation of amyloid structures as previous reported for highly modified albumin. A structural characterization of in vitro produced AGE-proteins will be useful to correctly interpret the pathophysiological significance of AGE products in diabetes.
血清蛋白糖化和晚期糖基化终产物（AGE）的形成与糖尿病并发症相关。高度糖基化的白蛋白常用于研究 AGE 蛋白的生化和细胞活性。然而，中度修饰的白蛋白样本预计在糖尿病研究中更具有（病理）生理学相关性。我们制备了一组九个中度修饰的白蛋白样本，并从侧链修饰、二级结构、折叠稳定性和光谱特性等方面对其进行了表征。
用葡萄糖、甲基乙二醛、乙醛酸和羧甲基赖氨酸对一组九个白蛋白样本进行了修饰，并从侧链修饰、热折叠稳定性、二级结构、聚集、表面电荷和配体结合等方面进行了表征。所采用的分析工具包括化学分析、侧链修饰的生化和免疫化学分析、近紫外圆二色性、差示扫描量热法、分析尺寸排阻和离子交换 HPLC，以及配体结合分析。
血清白蛋白的中度糖化和 AGE 修饰会导致结构变化，这取决于修饰试剂的化学反应性和用于体外糖化的浓度。一般来说，α-螺旋含量降低，热变性行为发生改变。然而，与先前报道的高度修饰的白蛋白不同，中度糖化不会引起聚集或形成淀粉样结构。对体外产生的 AGE 蛋白进行结构表征，将有助于正确解释 AGE 产物在糖尿病中的病理生理意义。
