Aggregation behavior of casein is correlated with the type of glycation-inducing agent

• Different glycation-inducing agents exert distinct effects on casein.
• Methylglyoxal-treated casein shows a higher level of advanced glycation end-products.
• Amyloid-like fibrillar species are present in methylglyoxal-treated casein samples.
• Methylglyoxal displays a greater reactivity toward casein as compared with ribose.

It has been discovered that protein glycation is involved in several human degenerative diseases and/or disorders, such as diabetes and Alzheimer's disease. Evidence suggests that protein glycation results in the formation of fructosamine and advanced glycation end-products (AGEs), accompanied by the alteration of protein conformation. To investigate how casein is influenced by glycation induced by ribose or methylglyoxal, structural characterization was carried out using thioflavin T (ThT) binding assay, far-UV circular dichroism, intrinsic fluorescence spectroscopy, light scattering, turbidity measurement, and transmission electron microscopy. AGE-related fluorescence measurement and nitro blue tetrazolium assay were used to examine the information regarding the pathway and relevant products associated with glycation. Degree of modification was characterized by determining the contents of lysine and arginine residues. We found that treatment of casein with glycation-inducing agents/glycation modifiers led to an obvious structural unfolding and enhanced solvent-exposed hydrophobic regions. In addition, we found that the concentration profile and production rate of fructosamine and/or AGEs were dependent on the type of glycation-inducing agent used. Moreover, our results showed that amyloid/amyloid-like aggregated species, with relative higher β-sheet secondary structure content and ThT fluorescence-positive characteristics, were found only when casein was treated with methylglyoxal. Finally, modification with methylglyoxal resulted in a higher percentage of reacted lysine or arginine residue in the casein sample. We believe the results of this work could aid in understanding the process of protein glycation.