Shamsi Anas, Amani Samreen, Alam Md Tauqir, Naeem Aabgeena
Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, 202002, India.
Eur Biophys J. 2016 Sep;45(6):523-34. doi: 10.1007/s00249-016-1119-0. Epub 2016 Mar 26.
Advanced glycation end products (AGEs) as a result of Maillard reaction are currently at the heart of the pathogenesis of several diseases and hence are the objective of numerous investigations. Glycation of proteins has been an implication in long-term complications. Collagen is the most abundant protein in the human body. The purpose of this study was to monitor and characterize the oligomeric aggregates and AGEs of human collagen on addition of glyoxal using ultraviolet, fluorescence, circular dichroism (CD) spectroscopy, docking studies, ITC, and microscopy. Collagen was incubated for varying time periods up to 21 days with three different concentrations (5, 20, and 40 mM) of glyoxal. Collagen exists as molten globule at day 6, evident from native-like secondary structure, altered tryptophan, and high ANS fluorescence due to surface-exposed hydrophobic residues. Glycated collagen as AGEs and aggregates was observed at day 18 and 21, respectively. Formation of AGE and aggregates were confirmed by UV and fluorescence spectroscopy. The obtained AGEs were characterized with respect to the extent of side chain modifications (lysine and arginine) forming the Schiff base, the carboxymethyl lysine, and carbonyl content. Non-tryptophan fluorescence for AGEs was also monitored as the emission peak at 400 and 440 nm, respectively. SEM and TEM confirmed the oligomeric nature of aggregates. Glyoxal at 40 mM shows maximum alterations in protein structure followed by 20 and 5 mM concentration. In the present paper, we propose that a high concentration of glyoxal for a prolonged time results in the formation of harmful aggregates and AGEs.
美拉德反应产生的晚期糖基化终末产物（AGEs）是目前多种疾病发病机制的核心，因此也是众多研究的对象。蛋白质糖基化与长期并发症有关。胶原蛋白是人体中含量最丰富的蛋白质。本研究的目的是利用紫外、荧光、圆二色性（CD）光谱、对接研究、等温滴定量热法（ITC）和显微镜技术，监测并表征添加乙二醛后人胶原蛋白的寡聚聚集体和AGEs。胶原蛋白与三种不同浓度（5、20和40 mM）的乙二醛孵育不同时间段，长达21天。在第6天，胶原蛋白以熔球状态存在，这从类似天然的二级结构、色氨酸改变以及由于表面暴露的疏水残基导致的高ANS荧光可以看出。分别在第18天和第21天观察到了作为AGEs和聚集体的糖化胶原蛋白。通过紫外和荧光光谱证实了AGEs和聚集体的形成。对所获得的AGEs进行了表征，涉及形成席夫碱的侧链修饰（赖氨酸和精氨酸）程度、羧甲基赖氨酸和羰基含量。还监测了AGEs的非色氨酸荧光，其发射峰分别在400和440 nm处。扫描电子显微镜（SEM）和透射电子显微镜（TEM）证实了聚集体的寡聚性质。40 mM的乙二醛对蛋白质结构的改变最大，其次是20 mM和5 mM浓度。在本文中，我们提出长时间高浓度的乙二醛会导致有害聚集体和AGEs的形成。
