New clevable photoreactive heterobifunctional cross-linking reagents for studying membrane organization.
The synthesis is described of four new cleavable, photosensitive, heterobifunctional cross-linking reagents for use in examining membrane organization: 4'-azidoazobenzene-4oxysuccinimide ester (1), N-[4-(p-azidophenylazo)benzoyl]-3-aminopropyl-N'-oxysuccinimide ester (2), N-[4-(p-azidophenylazo)benzoyl]-6-aminohexyl-N'-oxysuccinimide ester (3), and N-[4-(p-azidophenylazo)-benzoyl]-11-aminoundecyl-N'-oxysuccinimide ester (4). Two photoaffinity-directed cross-linking agents were prepared by attaching reagents 1 and 2 via their activated ester groups to soybean agglutinin (subunit Mr 30 000). Irradiation of the lectin derivatives resulted in a decrease in their absorption spectra at 360 nm due to photolysis of the bound reagents. Cross-linking of soybean agglutinin subunits following irradiation of the soybean agglutinin derivative to which reagent 2 had been coupled was observed by the appearance of new Coomassie blue staining material (60 000, 90 000, and 120 000 daltons) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On polyacrylamide gel electrophoresis in the absence of sodium dodecyl sulfate, soybean agglutinin oligomers were observed. Cleavage of the cross-linked soybean agglutinin with 0.1 M sodium dithionite for 25 min at room temperature resulted in the disappearance of the high molecular weight bands and an increase in the amount of uncross-linked material. The use of the photoaffinity-directed agents probing membrane organization is discussed.
D001386:Azides / Q000138:chemical synthesis*; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D037102:Lectins; D009682:Magnetic Resonance Spectroscopy; D008566:Membranes / Q000648:ultrastructure*; D037121:Plant Lectins; D011485:Protein Binding; D013025:Glycine max; D013053:Spectrophotometry
D001386:Azides; D003432:Cross-Linking Reagents; D037102:Lectins; D037121:Plant Lectins; C026851:N-(4-(4-azidophenylazo)benzoyl)-3-aminopropyl-N'-oxysuccinimide ester; C026852:4'-azidoazobenzene-4-oxysuccinimide ester; C026850:N-(4-(4-azidophenylazo)benzoyl)-6-aminohexyl-N'-oxysuccinimide ester; C026849:N-(4-(4-azidophenylazo)benzoyl)-11-aminoundecyl-N'-oxysuccinimide ester