Glycation involves the non-enzymatic addition of reducing sugars and/or their reactive degradation products to amine groups on proteins. This process is promoted by the presence of elevated blood glucose concentrations in diabetes and occurs with various proteins that include human serum albumin (HSA). This review examines work that has been conducted in the study and analysis of glycated HSA. The general structure and properties of HSA are discussed, along with the reactions that can lead to modification of this protein during glycation. The use of glycated HSA as a short-to-intermediate term marker for glycemic control in diabetes is examined, and approaches that have been utilized for measuring glycated HSA are summarized. Structural studies of glycated HSA are reviewed, as acquired for both in vivo and in vitro glycated HSA, along with data that have been obtained on the rate and thermodynamics of HSA glycation. In addition, this review considers various studies that have investigated the effects of glycation on the binding of HSA with drugs, fatty acids and other solutes and the potential clinical significance of these effects.

Background-Non-enzymatic glycation of human serum albumin (HSA) is associated with the long-term complications of diabetes. We examined the structure and location of modifications on minimally glycated HSA and considered their possible impact on the binding of drugs to this protein.Methods-Minimally glycated and normal HSA (used as a control) were digested with trypsin, Glu-C or Lys-C, followed by fractionation of the resulting peptides and their analysis by matrixassisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) to determine the structures and locations of glycation adducts.Results-Several specific lysine and arginine residues were identified as modification sites in minimally glycated HSA. Residues K12, K51, K199, K205, K439 and K538 were found to be modified through the formation of fructosyl-lysine, while the modification of K159 and K286 involved the formation of pyrraline, N ε -carboxymethyl-lysine respectively. Lysine K378 was found to give N ε -carboxyethyl-lysine in some forms of glycated HSA but fructosyl-lysine in other forms. Residues R160 and R472 produced a modification based on N ε -(5-hydro-4-imidazolon-2-yl) ornithine. Lysine R222 was modified to produce argpyrimidine, N ε -[5-(2,3,4-trihydroxybutyl)-5-hydro-4-imidazolon-2-yl]ornithine or tetrahydropyrimidine.Conclusions-With the exception of K12, K199, K378, K439 and K525, all of the observed sites of modification for minimally glycated HSA were new to this current study. The fact that many of these glycation-related modifications are located at or near known drug binding sites on HSA explains why some differences have been previously noted in the binding of certain drugs to normal vs glycated HSA.

Two techniques were developed for the immobilization of proteins and other ligands to silica through sulfhydryl groups. These methods made use of maleimide-activated silica (the SMCC method) or iodoacetyl-activated silica (the SIA method). The resulting supports were tested for use in high-performance affinity chromatography by employing human serum albumin (HSA) as a model protein. Studies with normal and iodoacetamide-modified HSA indicated that these methods had a high selectivity for sulfhydryl groups on this protein, which accounted for the coupling of 77-81% of this protein to maleimide- or iodoacetyl-activated silica. These supports were also evaluated in terms of their total protein content, binding capacity, specific activity, nonspecific binding, stability, and chiral selectivity for several test solutes. HSA columns prepared using maleimide-activated silica gave the best overall results for these properties when compared to HSA that had been immobilized to silica through the Schiff base method (i.e., an amine-based coupling technique). A key advantage of the supports developed in this work is that they offer the potential of giving greater site-selective immobilization and ligand activity than amine-based coupling methods. These features make these supports attractive in the development of protein columns for such applications as the study of biological interactions and chiral separations.

Background One of the long term complications of diabetes is the non-enzymatic addition of glucose to proteins in blood, such as human serum albumin (HSA), which leads to the formation of an Amadori product and advanced glycation end products (AGEs). This study uses 16O/18O-labeling and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to provide quantitative data on the extent of modification that occurs in the presence of glucose at various regions in the structure of minimally glycated HSA. Methods Normal HSA, with no significant levels of glycation, was digested by various proteolytic enzymes in the presence of water, while a similar sample containing in vitro glycated HSA was digested in 18O-enriched water. These samples were then mixed and the 16O/18O ratios were measured for peptides in each digest. The values obtained for the 16O/18O ratios of the detected peptides for the mixed sample were used to determine the degree of modification that occurred in various regions of glycated HSA. Results Peptides containing arginines 114, 81, or 218 and lysines 413, 432, 159, 212, or 323 were found to have 16O/18O ratios greater than a cut off value of 2.0 (i.e., a cut off value based on results noted when using only normal HSA as a reference). A qualitative comparison of the 16O- and 18O-labeled digests indicated that lysines 525 and 439 also had significant degrees of modification. The modifications that occurred at these sites were variations of fructosyl-lysine and AGEs which included 1-alkyl-2-formyl-3,4-glycoyl-pyrole, and pyrraline. Conclusions Peptides containing arginine 218 and lysines 212, 413, 432, and 439 contained high levels of modification and are also present near the major drug binding sites on HSA. This result is clinically relevant because it suggests the glycation of HSA may alter its ability to bind various drugs and small solutes in blood.

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