Change in the protein tertiary structure with non-enzymatic glycosylation of calf alpha-crystallin.

Non-enzymatic glycosylation of calf alpha-crystallin was induced by incubation with glucose. Glycosylated and non-glycosylated proteins were separated by affinity chromatography on Glyco Gel B boronic acid and were studied by circular dichroism (CD) and fluorescence. CD indicated that the glycosylated protein secondary structure was not altered, but the tertiary structure did undergo some changes. The CD band of this protein between 290 and 310 nm decreased in intensity. Extrinsic fluorescence probes, TNS (6-(p-toluidinyl) naphthalene-2-sulfonate) and MIANS (6-(4'-maleimidyl-anilino) naphthalene-2-sulfonic acid), indicated changes in both TNS binding sites and the sulfhydryl groups to a less hydrophobic microenvironment. Our results suggest that the glycosylation of protein leads to partial unfolding, and facilitates the sulfhydryl to oxidation.