Immunochemical Evidence Supporting 2-Pentylpyrrole Formation on Proteins Exposed to 4-Hydroxy-2-nonenal

Abstract

Previous model studies suggested the formation of lysine-based 2-pentylpyrroles as novel late adduction products formed upon exposure of proteins to the lipid peroxidation product 4-hydroxy-2-nonenal (HNE). Two 2-pentylpyrrole immunogens were prepared, one by treating keyhole limpet hemocyanin (KLH) directly with 4-oxononanal and the other by preformation of 6-(2-pentylpyrrol-1-yl)hexanoic acid from 6-aminocaproic acid and 4-oxononanal, followed by carbodiimide coupling to KLH. Pyrrole content and lysine modification in KLH were assayed independently. Following immunization of rabbits, antibody titer increased and plateaued over a 4 month period. The structural specificity of the IgG fractions of the antisera was evaluated through comprehensive competitive ELISA studies. These antibodies were used to verify the time-dependent appearance of the 2-pentylpyrrole epitope in protein exposed to HNE. Potential advantages of antibodies recognizing "advanced lipid peroxidation end products" over those recognizing "early" HNE adduction products are discussed.

Absence of cellular stress in brain after hypoxia induced by arousal from hibernation in Arctic ground squirrels

“…Prior studies showed that lipid peroxidation and protein oxidation occur mainly during the period of reperfusion (39), suggesting that oxidative stress incurred during arousal would be evident within the time course studied. CML is a rapidly formed, stable product of both lipid peroxidation and glycation processes (46), and HNE is a stable marker of lipid peroxidation (47). Absence of oxidative modification in brain is consistent with our previous studies showing that reduced glutathione and ascorbate are either maintained in brain throughout hibernation and arousal or increased slightly during late arousal (13,33,58).…”

Protein Adducts of Iso[4]levuglandin E2, a Product of the Isoprostane Pathway, in Oxidized Low Density Lipoprotein

“…HNE-HSA, NaCNBH 3 -reduced HNE-HSA, and MDA-HSA were prepared as described previously (38). ON-KLH antibodies (36) and LGE 2 -KLH antibodies (27) were prepared as described previously.…”

“…2-methylpyrrole, 4-oxopentanal-BSA (36), that lacks prostanoid or isoprostanoid side chains. The data presented in Table II establish that each of these antibodies shows outstandingly low cross-reactivity toward protein adducts of the structurally isomeric levulinaldehyde derivative.…”

Lipid Peroxidation Modification of Protein Generates Nϵ-(4-Oxononanoyl)lysine as a Pro-inflammatory Ligand

“…Conjugate-A standard KLH solution was prepared according to the published procedure (25), and the protein concentration (7.35 mg/ml) was determined by the bicinchoninic acid (BCA) assay using bovine serum albumin (BSA) as the standard. To the standard KLH solution (2.7 ml, 19.8 mg) was added 2.3 ml of doubly distilled water and 4-oxononanoic acid N-hydroxysulfosuccinimide ester (16.3 mg, 0.044 mmol).…”