Moderate glycation of serum albumin affects folding, stability, and ligand binding.

Serum protein glycation and formation of advanced glycation endproducts (AGE) correlates with diabetic complications. Highly AGE-modified albumin is frequently used to study the biochemical and cellular activities of AGE-proteins. However, moderately modified albumin samples are expected to be (patho)physiologically more relevant for diabetes research. We produced a panel of nine moderately modified albumin samples and characterized these in terms of side chain modifications, secondary structure, folding stability, and spectroscopic properties. A panel of nine albumin samples modified with glucose, methylglyoxal, glyoxylic acid and carboxymethyl lysine was characterized in terms of side chain modifications, thermal folding stability, secondary structure, aggregation, surface charge, and ligand binding. The analytical tools employed included chemical analysis, biochemical and immunochemical assays for side chain modifications, near UV circular dichroism, differential scanning calorimetry, analytical size exclusion and ion exchange HPLC, and a ligand binding assay. Moderate glycation and AGE modification of serum albumin causes structural changes that depend on the chemical reactivity of the modifying reagent and the concentration used for in-vitro glycation. In general, the α-helical content is decreased and thermal unfolding behavior is altered. However, moderate glycation does not cause aggregation or formation of amyloid structures as previous reported for highly modified albumin. A structural characterization of in vitro produced AGE-proteins will be useful to correctly interpret the pathophysiological significance of AGE products in diabetes.