The milieu within pancreatic β cells represents a favorable environment for glycation of insulin. Therefore, in this study, insulin samples were individually subjected to glycation under reducing and nonreducing conditions. As monitored by ortho-phthalaldehyde and fluorescamine assays, the reduced glycated insulin adduct demonstrates extensively higher level of glycation than the nonreduced glycated counterpart. Also, gel electrophoresis experiments suggest a significant impact of glycation under a reducing system on the level of insulin oligomerization. Furthermore, reduced and nonreduced glycated insulin adducts respectively exhibit full and partial resistance against dithiothreitol-induced aggregation. The results of thioflavin T and Congo red assays suggest the existence of a significant quantity of amyloid-like entities in the sample of reduced glycated insulin adduct. Both fluorescence and far-ultraviolet circular dichroism studies respectively suggest that the extents of unfolding and secondary structural alteration were closely correlated to the level of insulin glycation. Moreover, the surface tension of two glycated insulin adducts was inversely correlated to their glycation extents and to the quantity of exposed hydrophobic patches. Overall, the glucose-modified insulin molecules under reducing and nonreducing systems display different structural features having significant consequences on aggregation behaviors and surface tension properties. The particular structural constraints of glycated insulin may reduce the binding interaction of this hormone to its receptor which is important for both insulin function and clearance.

Total soluble lens proteins (TSPs) and α-crystallin (α-Cry) were individually subjected to the long-term glycation in the presence of d-glucose. The glycated and non-glycated protein counterparts were incubated under different stress conditions and compared according to their structure, stability and aggregation propensity by various spectroscopic techniques and gel mobility shift analyses. Extensive glycation of the lens proteins was accompanied with structural alteration, reduction in their surface hydrophobicity and increment of their surface tension. Our results suggest that glycation causes lens crystallins to partially resist against structural alteration and aggregation/fibrillation under both thermal and thermochemical systems. The conformational stability of lens crystallins was increased upon glycation, showing the reason behind resistance of glycated proteins against stress-induced structural alteration and aggregation. Due to the resistance of glycated lens crystallins against aggregation, the role of this modification in development of senile cataract can be explained with the associated damaging consequences highlighted in this article.

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