New Evidence for the Diversity of Mechanisms and Protonated Schiff Bases Formed in the Non-Enzymatic Covalent Protein Modification (NECPM) of HbA by the Hydrate and Aldehydic Forms of Acetaldehyde and Glyceraldehyde.

Acetaldehyde is a physiological species existing in blood. Glyceraldehyde is a commonly-used surrogate for glucose in studies of nonenzymatic glycation. Both species exist in dynamic equilibrium between two forms, an aldehyde and a hydrate. Nonenzymatic covalent protein modification (NECPM) is a process whereby a protein is covalently modified by a non-glucose species. The purpose here was to elucidate the NECPM mechanism(s) for acetaldehyde and glyceraldehyde with human hemoglobin (HbA). For the first time, both aldehydic and hydrate forms of acetaldehyde and glyceraldehyde were considered. Computations and model reactions followed by 1H NMR were employed. Results demonstrated that the aldehyde and hydrate forms of acetaldehyde bind and covalently-modify Val1 of HbA via different chemical mechanisms, yet generated an identical protonated Schiff base (PSB). The aldehyde and hydrate of glyceraldehyde also covalently-modified Val1 via mechanisms distinct from one another, yet generated an identical PSB. It is noteworthy that the PSB from acetaldehyde and glyceraldehyde were different structures. The PSB from acetaldehyde is proposed to proceed to covalent adducts that have been implicated in alcohol toxicity. Conversely, the PSB generated from glyceraldehyde can form an Amadori which has been implicated in diabetic complications. Thus, the PSB structure generated from acetaldehyde versus glyceraldehyde may be central to pathophysiological outcomes because it determines the structure of the stable covalent adduct formed.

Potential roles of inorganic phosphate on the progression of initially bound glucopyranose toward the nonenzymatic glycation of human hemoglobin: mechanistic diversity and impacts on site selectivity.

Nonenzymatic glycation (NEG) begins with the non-covalent binding of a glucopyranose to a protein. The bound glucopyranose must then undergo structural modification to generate a bound electrophile that can reversibly form a Schiff base, which can then lead to Amadori intermediates, and ultimately to glycated proteins. Inorganic phosphate (Pi) is known to accelerate the glycation of human hemoglobin (HbA), although the specific mechanism(s) of Pi as an effector reagent have not been determined. The aim of this study was to determine whether Pi and a glucopyranose can concomitantly bind to HbA and react while bound within the early, noncovalent stages to generate electrophilic species capable of progress in NEG. 31P and 1HNMR of model reactions confirm that bimolecular reactions between Pi and glucopyranose occur generating modified glucose electrophiles. Computations of protein/substrate interactions predict that Pi can concomitantly bind with a glucopyranose in HbA pockets with geometries suitable for multiple acid/base mechanisms that can generate any of four transient electrophiles. Pi-facilitated mechanisms in the noncovalent stages predict that the glycation of ß-Val1 of HbA to HbA1c is a "hot spot" because the ß-Val1 pocket facilitates many more mechanisms than any other site. The mechanistic diversity of the Pi effect within the early noncovalent stages of NEG predicts well the overall site selectivity observed from the in vivo glycation of HbA in the presence of Pi. These insights extend our basic understanding of the NEG process and may have clinical implications for diabetes mellitus and even normal aging.