Prolonged Incubation with Glucose Induces Oligomerization and Aggregation of Hemoglobin: Role of Advanced Glycation End Product on Protein Structure

Non-enzymatic glycation is a post-translational modification of proteins that leads to protein crosslinking, aggregation, conformational changes, etc. Chemical reaction of sugars with proteins (Maillard reaction) leads to formation of glycated adducts, known as Advanced Glycation End Products (AGE). HbA1c, the major glycated hemoglobin, increases proportionately with blood glucose concentration in diabetes mellitus. Glycation-induced modification of hemoglobin is a major cause of oxidative stress in diabetic patients. In this study, we have investigated the effect of different concentrations of glucose on hemoglobin following long-term incubation (30 days) with the heme protein. Gel electrophoretic profile revealed the presence of high molecular weight oligomers and aggregated species of hemoglobin following incubation with glucose. MALDI-MS analysis also indicated the appearance of high molecular weight oligomeric species of hemoglobin following modification with glucose. Secondary structural analysis showed that glucose induces a change in native secondary structure of the protein from α-helix to β-sheet, particularly cross-β structure. Scanning electron microscopic imaging studies revealed the presence of amorphous protein aggregates. Considering the increased level of glucose in diabetes mellitus, the current study appears clinically significant, particularly in the context of understanding glycation-mediated complications and AGE formation in proteins under physiological conditions.