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Biochemistry. 1980 Sep 16;19(19):4423-9.

New clevable photoreactive heterobifunctional cross-linking reagents for studying membrane organization.

Abstract

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.

PMID:
7190840
DOI:
10.1021/bi00560a007
[Indexed for MEDLINE]

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