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Determination of lysine modification product ε‐ N ‐pyrrolylnorleucine in hydrolyzed proteins and trout muscle microsomes by micellar electrokinetic capillary chromatography
Author(s) -
Zamora Rosario,
Navarro José L.,
Hidalgo Francisco J.
Publication year - 1995
Publication title -
lipids
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.601
H-Index - 120
eISSN - 1558-9307
pISSN - 0024-4201
DOI - 10.1007/bf02537020
Subject(s) - chemistry , chromatography , micellar electrokinetic chromatography , bovine serum albumin , capillary electrophoresis , lysine , hydrolysate , trout , derivatization , hydrolysis , microsome , amino acid , biochemistry , high performance liquid chromatography , in vitro , biology , fish <actinopterygii> , fishery
ε‐ N ‐Pyrrolylnorleucine (Pnl) is a product of the reaction between the lipid peroxidation product 4,5( E )‐epoxy‐2( E )‐heptenal (EH) and the ε‐amino group of lysine. Because Pnl might also be produced in proteins, a specific method to determine this compound in protein hydrolysates has been developed. Homoarginine, added as the internal standard, and Pnl are derivatized with diethyl ethoxymethylenemalonate and analyzed by micellar electrokinetic capillary chromatography. The method also analyzes lysine and arginine, and these analyses were useful in determining losses of these amino acids after treatment with EH. The lowest concentration of Pnl detected with acceptable reproductibility is 5 nmol/mL, and the coefficient of variation was determined from four standard curves assayed on separate days. Detector response was linear for samples containing 1.6 to 74 nmol/mL of Pnl. The assay was applied in investigations of Pnl production in bovine serum albumin (BSA) and trout muscle microsomes treated with EH. When BSA was incubated overnight with 30 mM EH, 76% of lysine residues were modified, and a part of these residues were detected as Pnl (12%). Pnl formation was also detected when trout muscle microsomes were incubated for three hours with 1 or 10 mM EH. These results show that Pnl is produced in vitro in proteins treated with the lipid peroxidation product EH, and suggest that Pnl might also be constituent of in vivo damaged proteins by their reaction with oxidized lipids.