Non-enzymatic protein glycosylation: back-titration assay

Abstract The glycosylation of albumin in vitro, as judged from the incorporation of d -[U- 14 C]glucose into trichloroacetic acid (TCA)-precipitable material represents a time-, temperature- and concentration-related process. It is markedly increased by NaCNBH 3 . A close correlation is observed between the radioactive data and the percentage of glycosylated albumin, as measured by affinity chromatography. The latter method, however, does not give information on the precise stoichiometry of the protein glycosylation. The relative extent of protein glycosylation can also be estimated by a back-titration procedure, the condensation of labelled d -glucose with the protein being inversely related to its prior degree of glycosylation. The back-titration assay was applied to plasma samples from normal and diabetic rats or human subjects and used to compare the glycosylation of albumin by distinct hexoses and hexose-phosphates. l -Glucose was found as efficient as d -glucose in causing albumin glycosylation and, hence, could conceivably be used to investigate in vivo changes in the intrinsic properties of extracellular proteins secondary to their glycosylation.