Effect of phosphate buffer on the kinetics of glycation of proteins

Abstract

The glycation of -globulin is catalyzed by phosphate buffer, whereas that of human serum albumin (HSA) and ovalbumin is not. The observed rate constant of spontaneous glycation of HSA is twofold larger than ovalbumin and -globulin. When D-glucose-2-h is compared with D-glucose-2-d, the overall kinetic isotope effect for the buffer-independent rates is H k 0 = D k 0 ¼ 4:43 AE 0:06 for HSA. The substrate isotope effect for the bufferindependent term excludes proton abstraction as the rate-determining step in the Amadori rearrangement by phosphate buffer. Catalysis by phosphate buffer of the glycation of -globulin indicates that phosphate is the abstracting base in the Amadori rearrangement. These results suggest that the phosphate buffer plays a fundamental role in the glycation of -globulin.

Stability of Protein Pharmaceuticals: An Update

“…Glycation has been seen for IgG2s (178), as well as IgG1s (177). Some buffer catalysis has been observed for glycation (179), at least with γ-globulin, with phosphate accelerating the reaction. However, buffer catalysis was not seen with BSA or ovalbumin.…”

Glycation and Serum Albumin Infiltration Contribute to the Structural Degeneration of Bioprosthetic Heart Valves

“…Non-cellular calcium-binding protein adsorption has been proposed in the context of BHV calcifications based on this reasoning 23 . Therefore, in this study, we hypothesized that glycation and infiltration by human serum albumin (HSA), the most abundant and glycation-susceptible circulating protein [24][25][26] , synergistically contribute to BHV SVD. Table I).…”

Effects of Buffer Composition on Site-Specific Glycation of Lysine Residues in Monoclonal Antibodies

“…We observed a larger increase in the rate of glycation for mAbs stressed in phosphate buffer (0.048 glc/Lys/d), consistent with previous observations. 15,16 The rate of glycation in sodium bicarbonate, on the other hand, was markedly higher (0.126 glc/Lys/d) than any other buffer across all mAbs studied.…”

“…Multiple publications have compared phosphate buffer to cationic buffers and have shown an increased rate of glycation for certain proteins incubated in phosphate. 15,16 This effect appears to be both protein and site-dependent, as specific lysine residues adjacent to known phosphate binding sites were shown to account for the rate enhancement. 10,16 Carbonate and arsenate have also been shown to increase the rate of glycation of specific proteins in vitro, 17 and certain food items have been shown to slow the rate of glycation in vivo.…”

“…During the candidate selection process, therapeutic candidate molecules are differentiated based on their susceptibility to various chemical liabilities, and this is often evaluated via stress-testing. Stress tests for glycation (i.e., forced glycation) have previously been conducted in a variety of buffers, 8,15,17,22 but in light of the fact that multiple reports have indicated that these buffer components can alter the site-specific rate of glycation, we sought to determine a buffer system for forced glycation experiments that most closely resembled the results seen in cell culture media, but with a drastically reduced chemical complexity (i.e., a single buffer component instead of the >20 that can be found in media), while also maintaining a high degree of site-specific variation between lysine residues to enable differentiation of candidate molecules. Here we describe the evaluation of multiple buffer systems for screening molecules by forced glycation in relation to glycation in cell culture media and provide a simple sequence-based hypothesis to explain some of the considerable variation we see in site-specific lysine glycation rates and patterns across various buffer systems.…”