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Changes in the fluorescence parameters of bound N-(1-pyrene) maleimide by lipid peroxidation of intestinal
T Ohyashiki1, T Yamamoto, T Mohri
1Department of Biochemistry, School of Pharmacy, Hokuriku University, Ishikawa, Japan.
Abstract:
Using a fluorogenic thiol reagent, N-(1-pyrene)maleimide (NPM), we have examined of lipid peroxidation on the microenvironment around SH groups of the membrane proteins in porcine intestinal brush-border membrane vesicles. The lipid peroxidation of the membranes was performed with various concentrations of t-butylhydroperoxide (t-BuOOH) in the presence of 100 microM ascorbic acid and 10 microM Fe2+. Treatment of NPM-labeled membranes with these oxidizing agents resulted in a decrease of the fluorescence lifetime, suggesting modification of the environmental properties around the bound dye. Measurement of the steady-state fluorescence anisotropy of the labeled membranes indicated restriction of the motion of the bound dye by the lipid peroxidation membranes. This interpretation was further supported by an elevation of the transition temperature of the anisotropy, a decrease in the quenching rate constant of the fluorescence with acrylamide and a decrease in the SH reactivity of the membrane proteins for NPM by lipid peroxidation. Based on these results, the possibility of conformation changes in the vicinity of SH groups in the membrane proteins associated with lipid peroxidation has been discussed.
Insights
Lipid peroxidation alters membrane protein microenvironments, affecting thiol group accessibility and protein conformation. This study used N-(1-pyrene)maleimide (NPM) to probe these changes in porcine intestinal brush-border membrane vesicles.
Area of Science:
- Biochemistry
- Membrane Biology
- Protein Chemistry
Background:
- Lipid peroxidation is a key process in oxidative stress.
- Membrane proteins play crucial roles in cellular functions.
- Understanding oxidative damage to membrane proteins is vital.
Purpose of the Study:
- To investigate the impact of lipid peroxidation on the microenvironment of SH groups in membrane proteins.
- To assess conformational changes in membrane proteins due to oxidative stress.
Main Methods:
- Utilized N-(1-pyrene)maleimide (NPM), a fluorogenic thiol reagent.
- Induced lipid peroxidation using t-butylhydroperoxide (t-BuOOH) with ascorbic acid and Fe2+.
- Measured fluorescence lifetime, anisotropy, and quenching kinetics.
Main Results:
- Lipid peroxidation decreased fluorescence lifetime and altered the microenvironment around SH groups.
- Observed restricted motion of NPM dye and increased transition temperature.
- Demonstrated decreased SH reactivity and acrylamide quenching rate constants.
Conclusions:
- Lipid peroxidation significantly modifies the microenvironment of membrane protein SH groups.
- Evidence suggests conformation changes in membrane proteins near SH groups following lipid peroxidation.
- These findings contribute to understanding oxidative damage mechanisms in biological membranes.