Understanding the impact of the molecular crowding environment on the glyoxal-mediated glycation of hemoglobin

In diabetes, the level of glyoxal (GO), a highly reactive oxoaldehyde increases from its normal value. GO reacts with various proteins and produces advanced glycated end products (AGEs). Living cells contain densely packed biomolecules, which exert crowding effects that may take up 20-40% of the entire volume of a cell. Molecular crowding significantly influences various cellular processes by affecting the behavior of molecules within the crowded intracellular environment, impacting protein folding, stability, interactions, and ultimately, the function of many biological pathways. Thus, it essentially acts as a key regulator of cellular activity within a confined space. The effect of the molecular crowding environment on the process of hemoglobin (Hb) glycation was studied in this work. Polyethylene glycol 200 (PEG 200) was used as a crowding agent. To execute the study, Hb was incubated with GO for a week at 37 °C in the presence of different percentages of PEG 200. Glycated Hb exhibited high absorbance at 280 nm, high turbidity, high browning, and a high melting temperature compared to the control Hb. Glycated Hb also showed higher thioflavin T (ThT) fluorescence and higher AGE fluorescence than the control Hb. Additionally, CD results indicated alterations in the secondary structures of glycated Hb. These glycation-mediated structural alterations of Hb can be reversed in a highly molecular crowded environment. A melting experiment performed on glycated Hb showed a small entropy change, indicating that the intermediate conformational transition is independent of entropic factors. Thus, the molecular crowding environment plays a significant role in preventing Hb from becoming glycated. This study may provide insight into the actual glycation scenario inside the human body.