Colorimetric assay of glycoprotein glycation free of interference from glycosylation residues

Abstract

We have developed a colorimetric assay for determining the degree of glycation of serum proteins that is unaffected by glycosylation residues. This was accomplished by reducing the proteins with sodium borohydride prior to periodate oxidation. Previous periodate-based methods, which offer several advantages over other glycation assays, cannot determine glycoprotein glycation because interference from sialic residues in the glycan chain can lead to overestimation of the amount of glycation products. Without reduction, glycation of fetuin was double that of asialofetuin glycated under identical conditions. We found that borohydride reduction before periodate oxidation increases the amount of formaldehyde released in proportion to the extent of glycation, irrespective of the degree of glycosylation. Using two glycoproteins and an unglycosylated protein, we showed how measurement of the formaldehyde increase enables the extent of glycoprotein glycation to be determined without removal of interfering sugars.