4-hydroxy-2-nonenal and sodium-cyanoborohydride

We developed a procedure to measure 4-hydroxy-2-nonenal (HNE)-amino acid adducts using the fluorescent probe 2-aminopyridine (2-AP). The method is based on the fact that HNE forms Michael addition-type amino acid adducts possessing an aldehyde functionality, which upon reaction with 2-AP in the presence of NaBH₃CN can be converted to their pyridylaminated derivatives. The HNE-amino acid adducts, namely Michael addition-type HNE-cysteine, HNE-histidine, and HNE-lysine adducts, after pyridylamination were resistant to conventional acid-hydrolysis conditions for protein (6N HCl/110°C/24 h) and could be detected by HPLC with a fluorescence detector. The reductive amination-based fluorescent labeling of HNE adducts is a simple and accurate technique that may be widely used to reveal increased levels of covalently modified proteins with HNE and its related aldehydes during aging and disease.

Topics: Aldehydes; Aminopyridines; Borohydrides; Chemistry Techniques, Analytical; Chromatography, High Pressure Liquid; Proteins; Spectrometry, Fluorescence

4-Hydroxy-2-nonenal (HNE), a major racemic product of lipid peroxidation, preferentially reacts with cysteine residues to form a stable HNE-cysteine Michael addition adduct possessing three chiral centers. Here, to gain more insight into sulfhydryl modification by HNE, we characterized the stereochemical configuration of the HNE-cysteine adducts and investigated their stereoselective formation in redox-regulated proteins. To characterize the HNE-cysteine adducts by NMR, the authentic (R)-HNE- and (S)-HNE-cysteine adducts were prepared by incubating N-acetylcysteine with each HNE enantiomer, both of which provided two peaks in reversed-phase high performance liquid chromatography (HPLC). The NMR analysis revealed that each peak was a mixture of anomeric isomers. In addition, mutarotation at the anomeric center was also observed in the analysis of the nuclear Overhauser effect. To analyze these adducts in proteins, we adapted a pyridylamination-based approach, using 2-aminopyridine in the presence of sodium cyanoborohydride, which enabled analyzing the individual (R)-HNE- and (S)-HNE-cysteine adducts by reversed-phase HPLC following acid hydrolysis. Using the pyridylamination method along with mass spectrometry, we characterized the stereoselective formation of the HNE-cysteine adducts in human thioredoxin and found that HNE preferentially modifies Cys(73) and, to the lesser extent, the active site Cys(32). More interestingly, the (R)-HNE- and (S)-HNE-cysteine adducts were almost equally formed at Cys(73), whereas Cys(32) exhibited a remarkable preference for the adduct formation with (R)-HNE. Finally, the utility of the method for the determination of the HNE-cysteine adducts was confirmed by an in vitro study using HeLa cells. The present results not only offer structural insight into sulfhydryl modification by lipid peroxidation products but also provide a platform for the chemical analysis of protein S-associated aldehydes in vitro and in vivo.

Topics: Aldehydes; Borohydrides; Chromatography, High Pressure Liquid; Cysteine; HeLa Cells; Humans; Lipid Peroxidation; Magnetic Resonance Spectroscopy; Mass Spectrometry; Models, Chemical; Oxidation-Reduction; Peptides; Proteins; Recombinant Proteins; Stereoisomerism