Post-translational modifications on glycated plasma fibrinogen: A physicochemical insight

Abstract

Methylglyoxal (MGO) is a highly reactive α-oxoaldehyde. It reacts with basic amino acids of the proteins to form advanced glycation end products (AGEs). Fibrinogen is a soluble multi-domain glycoprotein whose major function is to form fibrin clots that prevent blood loss upon vascular injury. In the present study, fibrinogen was incubated with varying concentration of MGO for 7 days followed by its biochemical and biophysical analysis. Glycated plasma fibrinogen (MGO-fibrinogen); exhibited hyperchromicity, a drop in tryptophan and intrinsic fluorescence, augmented AGE-specific fluorescence and melting temperature. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results showed decrease in mobility of MGO-fibrinogen. Structural perturbations in secondary and tertiary structure were identified by fourier transform-infrared spectroscopy (FT-IR), followed by far and near-UV circular dichroism (CD). Matrix-Assisted Laser Desorption Ionization–Time of Flight (MALDI-TOF-TOF) mass spectrometry studies suggested increase in molecular mass of MGO-fibrinogen. Amyloid like aggregates were confirmed by Thioflavin T (ThT), Congo red assay (CR), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The ketoamine moieties, carbonyl content (CO), hydroxymethylfurfural (HMF), superoxide and hydroxyl content were markedly elevated, whereas, total antioxidant capacity (TAC) and free thiol content decreased in MGO-fibrinogen as compared to the native protein. These investigations confirmed the structural and functional alterations in MGO-fibrinogen which leads to different physiological conditions like diabetes mellitus, cardiovascular disease etc.