New cleavable photoreactive heterobifunctional cross linking reagents for studying membrane organization

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Abstract
The synthesis is described of 4 new cleavable, photosensitive, heterobifunctional cross-linking reagents for use in examining membrane organization: 4''-azidoazobenzene-4-oxysuccinimide 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 MW 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 [SDS-PAGE]. On PAGE in the absence of SDS, 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 MW bands and an increase in the amount of uncross-linked material. The use of the photoaffinity-directed agents for probing membrane organization is discussed.