Kinetics of Nonenzymatic Glycation of Ribonuclease A Leading to Advanced Glycation End Products. Paradoxical Inhibition by Ribose Leads to Facile Isolation of Protein Intermediate for Rapid Post-Amadori Studies

Nonenzymatic glycation (Maillard reaction) of long-lived proteins is a major contributor to the pathology of diabetes and possibly aging and Alzheimer's disease. We report here kinetic studies of the glycation of the model protein ribonuclease A by glucose and ribose leading to the formation of antigenic advanced glycation end products (“AGEs”), detectable by AGE-specific polyclonal antibodies, and pentosidine, an acid-stable fluorescent AGE. As anticipated, the kinetics of glycation by ribose were considerably faster than by glucose, and the rate of AGE formation initially increased with increasing sugar concentrations. However, ribose above 0.15 M appeared to paradoxically slow the kinetics of AGE formation, suggesting ribose inhibits the conversion of “early” Amadori rearrangement products to “late” AGEs and thus favors the accumulation of reactive Amadori intermediates. The facile isolation of such protein intermediates was achieved by an “interrupted glycation” protocol in which free and reversibly bound (Schiff base) ribose was removed following a short (24 h) initial incubation with 0.5 M ribose at 37 °C. The kinetics of buildup of the Amadori intermediates and the kinetics of their post-Amadori conversion to antigenic AGEs were then independently studied. A rapid and reversible inhibition of the post-Amadori kinetics by free ribose was verified by direct re-addition of ribose to the isolated, sugar-free intermediate. The pH dependence of the kinetics of antigenic AGE formation from such intermediates was measured and exhibited an unusual bell-shaped profile over the pH range of 5.0−9.5 with a maximum near pH 8.0. Aminoguanidine, a pharmacological AGE inhibitor, was found to moderately or weakly inhibit antigenic AGE formation in such post-Amadori steps. The isolation of the glycated ribonuclease intermediate thus simplifies kinetic and mechanistic studies of AGE formation, permits AGE studies in the absence of complications arising from free or Schiff base bound sugar, and provides a novel methodology for evaluating the mechanism and efficacy of therapeutic agents that may inhibit AGE formation.