Rapid cross-linking of proteins by 4-ketoaldehydes and 4-hydroxy-2-alkenals does not arise from the lysine-derived monoalkylpyrroles

Exposure of proteins to 4-hydroxy-2-nonenal (HNE) results in conversion of lysines in part to 2-pentylpyrroles that can be formed in higher yield by e xposure to the isomeric 4-oxononanal. Since both HNE and 4-oxononanal cause protein cross-linking, and since pyrrolation of proteins by gamma-diketone s is also known to result in protein cross-linking, it has been considered that the initially formed 2-pentylpyrroles are responsible for the protein cross-linking seen for HNE and 4-oxononanal. Here we show that protein-boun d 2-alkylpyrrole products associated with modification by 4-hydroxy-2-alken als and 4-oxoalkanals, possessing only monoalkyl substitution, induce undet ectable levels of autoxidation-mediated protein cross-linking over time per iods where the parent aldehydes effect extensive protein cross-linking, whi ch then must be occurring through alternative mechanisms. Finally, using bo th RNase and BSA, our finding that reductive methylation of lysines blocks protein cross-linking induced by either HNE or 4-oxononanal (and developmen t of fluorescence in the case of HNE) implicates the obligatory role of lys ines in the cross-linking reactions.