Abstract
AimNon-enzymatic glycation impairs the structural and functional characterstics of human serum albumin (HSA) native conformation. Prolonged hyperglycemia causes cross links formation in proteins that may contribute to progression of diabetic complications. MethodsHSA (20μM) was incubated with different concentration of d-glucose100, 200, 300 and 400mg/dl for a period of 40 days in phosphate buffer saline (20mM pH=7.4) under sterile conditions. Incubated samples were extensively dialyzed and structural changes were analyzed by far and near UV circular dichroism spectra measurement. Fructosamine assay with nitroblue tetrazolonium was performed to confer isomerisation between glucose and protein. Aggregations of the glycated product (AGEs) formed during reduction of nitrobluetetrazolium dye were evaluated by transmission electron microscopy. Crosslinks aggregates were investigated by in-situ Congo red binding assay. Red blood cells hemolysis test was performed to decipher the antioxidant activity of albumin samples. ResultsFructosamine content in glycated albumin demonstrates the non-enzymatic addition of glucose to HSA and confers the formation of monoformazone (marker of glycation). Significant changes were found in the glycated samples of HSA compared to native (unmodified) in far and near UV circular dichroism. Transmission electron microscopy, Congo red staining, showed the formation of crosslink's aggregated mass in glycated HSA. Glycation of albumin reduces the antioxidant capacity of native albumin confirmed by red blood cells hemolysis test. ConclusionThe finding of present study brings new evidences on the detrimental alterations of on albumin vital functions after non-enzymatic glycation with glucose. These results may emphasize the albumin associated diabetic complications under glycemic range of diabetes mellitus.
Published Version
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