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The glycation (nonenzymatic glycosylation) of several proteins was studied in various buffiner os rder to assess the effects of buffering ions on the kinetics and specificity of glycation of protein. Incubation of RNase with glucose in phosphate buffer resulted in inactivation of the enzyme because of preferential modification of lysine residues ino r near the activsei te. In contrast, in the cationic buffers, 3-(N-morpholino)propanesulfonic acid and 3-(N-tris(hydroxymethyl)rnethylamino)- 2-hydroxypropanesulfonica cid, the kineticso f glycation of RNase were decreased 2- to 3-fold, there was a decrease in glycation of active site versus peripheral lysines, and the enzyme was resistant to inactivation by glucose. The extent of Schiff base formation on RNase was comparable in the three buffers, suggesting that phosphate, bound in the active site of RNase, catalyzed the Amadori rearrangement at active site lysines, leading to the enhanced rate of inactivation of the enzyme. Phosphate catalysis of glycation was concentration-dependent and could be mimicked by arsenate. Phosphate also stimulated the rate of glycation of other proteins, such as lysozyme, cytochrome c, albumin, and hemoglobin. As with RNase, phosphate affected the specificity of glycation of hemoglobin, resulting in increasegdly cation of amino-terminal valine versus intrachain lysine residues. 2,3-Diphosphoglycerate exerted similar effeocnt st he glycation of hemoglobin, suggesting that inorganic and organic phosphates may play an important role in determining the kinetics and specificity of glycation of hemoglobin in the red cell. Overall, these studies establishth at buffering ions or ligands can exert significant effects on the kinetics ands pecificity of glycation of proteins.


This research was originally published in Journal of Biological Chemistry. Watkins NG, Neglia-Fisher CI, Dyer DG, Thorpe SR, Baynes JW. Effect of Phosphate on the Kinetics and Specificity of Glycation of Protein. Journal of Biological Chemistry. 1987; 262:7207-7212. © the American Society for Biochemistry and Molecular Biology.

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