Changes in the fluorescence parameters of bound N-(1-pyrene)maleimide by lipid peroxidation of intestinal brush-border membranes

Abstract

Using a fluorogenic thiol reagent, N-(1-pyrene)maleimide (NPM), we have examined the effect 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-buthylhydroperoxide (t-BuOOH) in the presence of 100 μM ascorbic acid and 10 μM 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 of the 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.