Modification of Chloride Ion Transport in Human Erythrocyte Ghost Membranes by Photoaffinity Labeling Reagents Based on the Structure of 5-Nitro-2-(3-Phenylpropylamino)-benzoic Acid (NPPB)

Using human red blood cell ghost membranes, we have evaluated 5-nitro-2-[N-3-(4-azidophenyl)-propylamino]-benzoic acid and 5-nitro-2-[N-3-(4-azido-2,3,5,6-tetrafluorophenyl)-propylamino]-benzoic acid (FAzNPPB) as photoaffinity labeling agents based on the structure of the widely important Cl− channel blocker 5-nitro-2-(3-phenylpropyl-amino)-benzoic acid (NPPB). The tetrafluoro-substituted aryl azide was found to be a more effective photoinactivating agent than the corresponding protio compound. Using a tritiated version ([3H]FAzNPPB), we demonstrated that photoinactivation of Cl− flux was accompanied by photolabeling of the band 3 protein and membrane lipids. Both processes were diminished in the presence of NPPB and the related arylanthranilate flufenamic acid. Photolabeling resulted in the incorporation of 1.0 ± 0.2 mol 3H/mol protein in the band 3 integral membrane domain, whereas the cytoplasmic domain was essentially unlabeled, By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, photolabeling was found to be the result of partial labeling of at least three different regions of the membrane domain, Based on trypsin proteolysis, reverse-phase high-performance liquid chromatography and electrospray ionization mass spectrometry analysis, it is proposed that one of the sites of photolabeling is the peptide lys-phe-lys (590-592), FAzNPPB is a successful polyfluoro aryl azide photoaffinity labeling agent which may be of further use in studying the diverse effects of arylanthranilates on biological membranes.