Mechanism of formation and quantitation of imines, pyrroles, and stable nonpyrrole adducts in 2,5-hexanedione-treated protein.

The condensation of gamma-diketones with protein epsilon-amino moieties to yield alkylpyrrole adducts has been demonstrated in many in vitro and in vivo systems, although certain features of this reaction remain unclear. The present in vitro study was designed to examine additional aspects of gamma-diketone-protein interactions, including the possible formation of imine intermediates and stable nonpyrrole products, and the potential for conformational changes in pyrrolylated protein. Values for total, stable covalent binding were consistently higher than p-dimethylaminobenzaldehyde (DMAB)-detectable pyrrole adduct concentrations when bovine serum albumin (BSA) was incubated (24 hr, 37 degrees C) with [14C]-2,5-hexanedione (2,5-HD) at diketone:lysine ratios greater than or equal to 5:1 (at pH 9.5) or 1:1 (at pH 7.4). Treatment of pyrrolylated BSA with proteases before the DMAB assay decreased but did not eliminate the difference between these parameters. Quantitative amino acid analysis of pyrrolylated BSA revealed molar decreases in lysine content equivalent to DMAB-detectable pyrrole adduct concentrations; no other amino acids were significantly altered. Cleavage of disulfide bonds in pyrrolylated BSA by dithiothreitol resulted in an apparent decrease in DMAB-detectable pyrrole, which was reversible upon subsequent protease treatment. A similar decrease was not seen with pyrrolylated concanavalin A, a protein that lacks disulfide linkages. Samples of BSA were incubated with [14C]-2,5-hexanedione for 2-144 hr and a portion of each incubation mixture treated with NaCNBH3 to selectively reduce imines to stable amines. Substantial levels of an imine intermediate were detected at 2, 6, and 24 hr but not at 144 hr. The above findings support proposed mechanisms involving imine intermediates in the pyrrolylation reaction. In addition, evidence for the formation of stable nonpyrrole adducts at high diketone:amine molar ratios has been provided. Results consistent with potential conformational alterations in pyrrolylated protein have also been demonstrated.