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d -ribose, a reducing sugar, in diabetic hyperglycemia provokes non-enzymatic glycoxidation of hemoglobin (Hb), an abundant protein of red blood cells (RBCs). Different types of intermediates adduct formation occur during glycoxidation, such as advanced glycation end-products (AGEs) which lead to amyloid formation due to structural and conformational alterations in protein. Therefore, the study of these intermediate adducts plays a pivotal role to discern their relationship with diabetes mellitus and related disorders. Here, we investigated the interaction mechanism of d -ribose with Hb, and Hb prebound phytochemical thymoquinone (TQ). Our investigation reveals that the interaction of TQ with histidine residues of Hb interferes with the interaction of d -ribose with glycine residues at the glycation-site. Based on that, we had performed a time-based (21-days) in-vitro glycoxidation study at 37 °C to investigate the structural perturbation mechanism of Hb at different time-intervals in absence/presence of TQ. We found that prolonged glycoxidation induces amyloid formation in absence of TQ but in its presence, the process was prohibited. In summary, this study examined and characterized biophysically different intermediate-states of protein carrying glycoxidation-modification. Our findings suggested that TQ potentially affects interaction of d -ribose with Hb that prevents glycoxidation and protofibril formation, which establishes TQ as a potential therapeutic agent.

Non-enzymatic glycation is the addition of a free carbonyl group of a reducing sugar to the free amino groups of proteins, which results in the formation of early and advanced glycation end-products (AGEs). Glycation reaction is profoundly associated with diabetes and its secondary complications, such as nephropathy and neuropathy. Glyoxal is a carbonyl species that reacts rapidly with the free amino groups of proteins to form AGEs. While the formation of AGEs with various glycating agents has previously been demonstrated, no extensive studies have been conducted to assess the role of quercetin in all three stages of glycation (early, intermediate and late). In this study, we report the glycation of HSA (human serum albumin) and its characterization by several spectroscopic techniques. Furthermore, inhibition of products at all stages of glycation was studied by various assays. Spectroscopic analysis suggests structural perturbations in the HSA macromolecule as a result of modification, which might be due to the generation of free radicals and the formation of AGEs. Inhibition in the formation of glycation has established that quercetin is a better and a more potent antiglycating agent than aminoguanidine at all stages of glycation.

Nonenzymatic glycosylation of proteins finally generates advanced glycation end products (AGEs). The Schiff's base and Amadori adduct are stages of early glycation. AGE-modified IgG may undergo conformational alterations and the final entity of the process may be involved in the pathogenesis of Rheumatoid Arthritis (RA). In this study, glycation of human IgG was carried out with varying concentrations of glucose. Effect of incubation period on glycation of IgG has also been studied. Amadori adduct was detected by nitroblue tetrazolium (NBT) dye. The glucose mediated structural alterations in IgG were studied by UV, fluorescence, CD, FT-IR, DLS and DSC spectroscopy, and SDS-PAGE. Glycation-induced aggregation in AGE-IgG was reported in the form of binding of thioflavin T and congo red. Furthermore, AGE-modified IgG exhibited hyperchromicity, decrease of tryptophan fluorescence accompanied by increase in AGE specific fluorescence, loss of β-sheet, appearance of new peak in FT-IR, increase in hydrodynamic size and melting temperature. SDS-PAGE results showed decrease in the band intensity of glycosylated-IgG compared to native IgG. Glycation-induced modifications and aggregation of IgG might be important in the pathogenesis of RA.

Reactions of reducing sugars with free amino groups of proteins can form advanced glycation end products (AGEs). While the formation of nucleoside AGEs has been studied in detail, no extensive work has been carried out to assess DNA Amadori and DNA advanced glycation end products. In this study, we report biophysical/chemical characterization of glucose-induced changes in DNA, as well as DNA Amadori and DNA advanced glycation end products. Glucose treated DNA exhibited hyperchromicity, decrease in melting temperature, and enhanced emission intensity in a time dependent manner. Formation of DNA Amadori product and DNA advanced glycation end products, mainly CEdG ( N 2 -carboxyethyl-2′-deoxyguanosine), were the major outcome of the study.