Abstract
Lipid hydroperoxide may react with protein or amino phospholipid without secondary decomposition. We prepared a polyclonal antibody to lipid hydroperoxide-modified proteins using 13S-hydroperoxy-9Z, 11E-octadecadienoic acid-modified keyhole limpet hemocyanin (13-HPODE-KLH) as immunogen. The antibody recognized 13-HPODE-modified bovine serum albumin (BSA), but not aldehyde-modified proteins, such as malondialdehyde-modified BSA. The antibody also recognized adducts derived from 13-HPODE and 13S-hydroperoxy-9Z, 11E, 15Z-octadecatrienoic acid (13-HPOTRE(alpha)). The oxidized alpha-linolenic acid- and linoleate-protein adducts were recognized by the antibody. Oxidized phospholipid-protein adducts were scarcely recognized by the antibody. However, when ester bonds of phospholipids containing linoleic acid were hydrolyzed by alkaline treatment, the cross-reactivities appeared. The result suggests that a phospholipid hydroperoxide can react with a protein directly or indirectly, and a carboxyl terminal (COOH) of the lipid in an adduct was needed as an epitope. Oxidized LDL (ox-LDL) was prepared by the incubation of LDL with copper ion or 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), and the formation of lipid hydroperoxide-modified apolipoprotein was confirmed using the antibody. A slight immunoreactivity was observed in ox-LDL without alkaline treatment. When the ox-LDL was treated with alkali to hydrolyze the ester bonds of the lipid, enhanced antigenicity appeared with time-dependency. The results suggest that lipid hydroperoxide-modified apolipoprotein was formed during the oxidation of LDL.
Highlights
Lipid hydroperoxide may react with protein or amino phospholipid without secondary decomposition
The purified 13-HPODE was incubated at 37°C for 3 days with keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA) in phosphate buffer.The character of the lipid hydroperoxide-modified proteins was first investigated
The results assume that phospholipid hydroperoxides were covalently attached to BSA and that a cleavage of an ester bond is required for the formation of an antigenicity of oxidized phospholipid-modified protein
Summary
Lipid hydroperoxide may react with protein or amino phospholipid without secondary decomposition. A novel polyclonal antibody to lipid hydroperoxide-modified protein was prepared using 13S-hydroperoxy-SZ, 11E-octadecadienoic acid (13-HPODE)-modified keyhole limpet hemocyanin (KLH) as the immunogen. The isolation of oxidized lipid-modified BSA was Preparation of immunogen and anti-13-HPODE-KLH antibody performed by ethanol precipitation. The 13-HPODE-modified BSA was prepared by the incubation of lipid-free BSA (5 mg/ml) with 13-HPODE (5 mM) in 0.1 M phosphate buffer (pH 7.4) at 37°C for 3 days, followed by dialysis against PBS.
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