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Pyrrolization and antioxidant function of proteins following oxidative stress
F J Hidalgo1, M Alaiz, R Zamora
1Instituto de la Grasa, Consejo Superior de Investigaciones Científicas, Avenida Padre García Tejero 4, 41012-Sevilla, Spain.
Abstract:
The consequences of oxidative stress on microsomal proteins were analyzed by studying their pyrrolization and the antioxidative activity of the modified proteins produced. The microsomal system consisted of freshly prepared trout muscle microsomes, which were oxidized in the presence of 5 microM Cu(2+), 1 mM Fe(3+)/5 mM ascorbate, or 1 mM Cu(2+)/10 mM H(2)O(2). Pyrroles on proteins were detected by forming Ehrlich adducts with p-(dimethylamino)benzaldehyde and by determination of epsilon-N-pyrrolylnorleucine (Pnl) by capillary electrophoresis. Their antioxidative activity was studied by testing two model pyrrolized proteins (dimeric and monomeric modified bovine serum albumin: DBSA and MBSA, respectively), which were produced in the reaction of BSA and 4,5(E)-epoxy-2(E)-heptenal. These proteins were assayed at a concentration of 10-40 microg/mL, which was selected because at this concentration both DBSA and MBSA had a concentration of Pnl similar to the Pnl concentration produced in oxidized microsomes. Both DBSA and MBSA significantly (p < 0.05) protected against lipid peroxidation, assessed by the formation of thiobarbituric acid reactive substances (TBARS), and protein damage, evaluated by amino acid analysis, for the three systems assayed, and this protection was always higher than that exhibited by BSA, which was used as control. The order of effectiveness was DBSA > MBSA > BSA and was parallel to the Pnl content in the assayed proteins. These results suggest that antioxidative activity of BSA may also be related to its ability to react with lipid oxidation products and to produce modified BSA with antioxidative activity. This mechanism may also be contributing to the antioxidative activity exhibited by many proteins.
Insights
Oxidative stress in trout microsomes leads to protein pyrrolization. Modified bovine serum albumin (BSA) with pyrrole content demonstrated significant antioxidative activity, protecting against lipid peroxidation and protein damage.
Area of Science:
- Biochemistry
- Food Science
- Oxidative Stress Research
Background:
- Oxidative stress is a significant factor in food spoilage and protein damage.
- Microsomal proteins are susceptible to oxidative modifications.
- Pyrrolization is a modification occurring in proteins under oxidative conditions.
Purpose of the Study:
- To analyze the consequences of oxidative stress on microsomal proteins.
- To investigate the antioxidative activity of pyrrolized proteins.
- To understand the role of protein modification in combating oxidative damage.
Main Methods:
- Oxidation of trout muscle microsomes using copper and iron catalysts with ascorbate or hydrogen peroxide.
- Detection of protein pyrroles using Ehrlich adducts and capillary electrophoresis to quantify epsilon-N-pyrrolylnorleucine (Pnl).
- Assay of modified bovine serum albumin (BSA) – dimeric (DBSA) and monomeric (MBSA) – for antioxidative activity against lipid peroxidation (TBARS) and protein damage (amino acid analysis).
Main Results:
- Oxidized trout microsomes showed increased pyrrole formation.
- Both DBSA and MBSA significantly protected against lipid peroxidation and protein damage compared to native BSA.
- The protective effect correlated directly with the pyrrole content (Pnl) in the modified proteins, with DBSA > MBSA > BSA.
Conclusions:
- Protein pyrrolization, induced by oxidative stress, can generate modified proteins with significant antioxidative properties.
- Modified BSA exhibits enhanced protection against oxidative damage, suggesting a mechanism for protein's role in mitigating lipid peroxidation.
- This mechanism of pyrrolization and subsequent antioxidative activity may contribute to the overall protective functions of proteins in biological systems and food matrices.