Department of Biochemistry, Jawaharlal Nehru Medical College, Faculty of Medicine, Aligarh Muslim University, Aligarh, Uttar Pradesh, India.
Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India.
Spectrochim Acta A Mol Biomol Spectrosc. 2024 Feb 15;307:123500. doi: 10.1016/j.saa.2023.123500. Epub 2023 Oct 31.
Methylglyoxal (MGO); a reducing sugar and a dicarbonyl; attaches to the biomolecules (proteins, lipids, and DNA) leading to glycation and accumulation of oxidative stress in cells and tissues. Superoxide anion formed under such conditions entraps free nitric oxide radical (NO) to form peroxynitrite (PON). Nitro-oxidative stress due to PON is well established. Human fibrinogen plays a key role in haemostasis and is a highly vulnerable target for oxidation. Modifications of fibrinogen can potentially disrupt its structure and function. Earlier evidence suggested that glycation and nitro-oxidation lead to protein aggregation by making it resistant to lysis. This study aims to reveal the structural perturbations on fibrinogen in the presence of MGO and PON synergistically. The in vitro glyco-nitro-oxidation of human fibrinogen by MGO and PON leads to substantial structural alterations, as evident by biophysical and biochemical studies. In-silico results revealed the formation of stable complexes. UV-visible, intrinsic fluorescence, and circular dichroism investigations confirmed the synergistic effect of MGO and PON caused micro-structural modifications leading to secondary structural alterations. AGEs formation in MGO-modified fibrinogen reduced the free lysine and free arginine residues which were quantified by TNBS and phenanthrenequinone assays. Enhanced oxidative status was confirmed by estimating carbonyl content. ANS fluorophore validated exposure of hydrophobic patches in modified protein and thioflavin-T showed maximum binding with synergistically modified fibrinogen, indicated the formation of β-sheet. Confocal and electron microscope results corroborated the formation of aggregates. This study, therefore, evaluated the impact of MGO and PON on the structural integrity, oxidative status and aggregate formation of fibrinogen that can aggravate metabolic complications.
甲基乙二醛（MGO）；一种还原糖和二羰基化合物；与生物分子（蛋白质、脂质和 DNA）结合，导致糖化和细胞和组织中氧化应激的积累。在这种情况下形成的超氧阴离子自由基会捕获游离的一氧化氮自由基（NO）形成过氧亚硝酸盐（PON）。PON 引起的硝基氧化应激已得到充分证实。人纤维蛋白原在止血中起关键作用，是氧化的高度易损靶标。纤维蛋白原的修饰可能会破坏其结构和功能。早期的证据表明，糖化和硝基氧化导致蛋白质聚集，使其不易溶解。本研究旨在揭示 MGO 和 PON 协同作用下纤维蛋白原的结构扰动。体外实验表明，MGO 和 PON 对人纤维蛋白原的糖基化-硝基氧化导致了大量的结构改变，这可以通过生物物理和生化研究来证明。计算机模拟结果显示形成了稳定的复合物。紫外可见、内源荧光和圆二色性研究证实了 MGO 和 PON 的协同作用导致微结构修饰，从而导致二级结构改变。AGEs 在 MGO 修饰的纤维蛋白原中的形成减少了游离赖氨酸和游离精氨酸残基，这通过 TNBS 和菲咯啉醌测定来定量。通过估计羰基含量证实了增强的氧化状态。ANS 荧光团验证了修饰蛋白中疏水性斑点的暴露，并且与协同修饰的纤维蛋白原的最大结合表明形成了β-折叠。共聚焦和电子显微镜结果证实了聚集体的形成。因此，本研究评估了 MGO 和 PON 对纤维蛋白原结构完整性、氧化状态和聚集体形成的影响，这可能会加重代谢并发症。
