New Cleavable 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-4-oxysuccinimide ester (1), N-[4-(β-azidophenylazo) benzoyl]-3-aminopropyl-N^--oxysuccinimide ester (2), N-[4-(p-azidophenylazo)benzoyl]-6-aminohexyl-N^--oxysuccinimide ester (3), and Ar-[4-(β-azidophenylazo)- benzoyl]-l l-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 M_r 30000). 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 (60000, 90000, and 120000 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 for probing membrane organization is discussed.