Structural perturbations induced by cumulative action of methylglyoxal and peroxynitrite on human fibrinogen: An in vitro and in silico approach.

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.