Glutamic and aminoadipic semialdehydes are the main carbonyl products of metal-catalyzed oxidation of proteins

Abstract

Metal-catalyzed oxidation results in loss of function and structural alteration of proteins. The oxidative process affects a variety of side amino acid groups, some of which are converted to carbonyl compounds. Spectrophotometric measurement of these moieties, after their reaction with 2,4-dinitrophenylhydrazine, is a simple, accurate technique that has been widely used to reveal increased levels of protein carbonyls in aging and disease. We have initiated studies aimed at elucidating the chemical nature of protein carbonyls. Methods based on gas chromatography/mass spectrometry with isotopic dilution were developed for the quantitation of glutamic and aminoadipic semialdehydes after their reduction to hydroxyaminovaleric and hydroxyaminocaproic acids. Analysis of model proteins oxidized in vitro by Cu2+/ascorbate revealed that these two compounds constitute the majority of protein carbonyls generated. Glutamic and aminoadipic semialdehydes were also detected in rat liver proteins, where they constitute approximately 60% of the total protein carbonyl value. Aminoadipic semialdehyde was also measured in protein extracts from HeLa cells, and its level increased as a consequence of oxidative stress to cell cultures. These results indicate that glutamic and aminoadipic semialdehydes are the main carbonyl products of metal-catalyzed oxidation of proteins, and that this reaction is a major route leading to the generation of protein carbonyls in biological samples.

Figure 1

Reaction scheme for the formation of glutamic and aminoadipic semialdehydes and their reduction products HAVA and HACA.

Figure 2

Full scan chromatogram (A), mass spectrum (B), and fragmentation pattern of HAVA-TFAME derivative. (D) Mass spectrum of d5-HAVA-TFAME derivative.

Figure 3

Full scan chromatogram (A), mass spectrum (B), and fragmentation pattern of HACA-TFAME derivative. (D) Mass spectrum of d4-HAVA-TFAME derivative.

Figure 4

Chromatograms of HAVA (A) and HACA (B) standards, prepared as described in Materials and Methods. OPA derivatives of samples were prepared automatically and derivatized samples analyzed by RP-HPLC.

Figure 5

Analysis of HAVA and HACA in an oxidized GS sample. The sample was reduced, spiked with d5-HAVA and d4-HACA, hydrolyzed, and derivatized as described in Material and Methods. SIM traces with m/z = 280, 285, 294, and 298 correspond to HAVA, d5-HAVA spike, HACA, and d4-HACA spike, respectively.

Figure 6

Glutamic semialdehyde in HeLa cells subjected to oxidative stress by exposure to glucose oxidase/glucose. Cells were grown to confluency and then glucose oxidase/glucose added to the medium to generate the indicated concentration of H₂O₂ as determined analytically. Results are expressed as mmol/mol of an average 50-kDa protein. Each bar represents analysis of three independent protein samples from a single culture. Error bars are SD.