/sup 125/I-labeled crosslinking reagent that is hydrophilic, photoactivatable, and cleavable through an azo linkage
A radioactive crosslinking reagent, N-(4-(p-azido-m-(/sup 125/I)iodophenylazo)benzoyl)-3-aminopropyl-N'-oxysulfosuccinimide ester, has been synthesized. The reagent is photoactivatable, water-soluble, cleavable through an azo linkage, and labeled with /sup 125/I at the carrier-free specific activity of 2000 Ci/mmol. Any protein derivatized with the reagent is thus converted into an /sup 125/I-labeled photoaffinity probe. Crosslinks are formed following photolysis with 366-nm light, and cleavage by sodium dithionite results in the donation of radioactivity to the distal partner in crosslinked complexes. The newly labeled proteins are then analyzed by gel electrophoresis and autoradiography. The compound was prepared by iodination of N-(4-(p-aminophenylazo)benzoyl)-3-aminopropionic acid using carrier-free Na/sup 125/I and chloramine-T, followed by azide formation and conversion to the water-soluble sulfosuccinimide ester. As a model system, protein A-Sepharose was derivatized with the reagent under subdued light. Each derivatized protein A molecule contained only one crosslinker. The derivatized protein A-Sepharose was then photolyzed in the presence of human serum and subsequently treated with sodium dithionite. Analysis of the serum by gel electrophoresis revealed that 1.1% of the radioactive label originally present on the protein A-Sepharose was transferred to the heavy chain of IgG, which was the most intensely labeled protein in the gel. The next most intensely labeled protein was IgG light chain, which incorporated radioactivity that was lower by a factor of 3.6 than that of the heavy chain. 36 references, 3 figures.
- Research Organization:
- Rockefeller Univ., New York, NY
- OSTI ID:
- 5871040
- Journal Information:
- Proc. Natl. Acad. Sci. U.S.A.; (United States), Journal Name: Proc. Natl. Acad. Sci. U.S.A.; (United States) Vol. 81:21; ISSN PNASA
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
BETA DECAY RADIOISOTOPES
CARBOXYLIC ACIDS
CHEMICAL PREPARATION
CHEMICAL REACTIONS
CROSS-LINKING
DAYS LIVING RADIOISOTOPES
DECOMPOSITION
DICARBOXYLIC ACIDS
ELECTRON CAPTURE RADIOISOTOPES
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
LABELLED COMPOUNDS
LABELLING
MATHEMATICAL MODELS
NUCLEI
ODD-EVEN NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
PHOTOCHEMICAL REACTIONS
PHOTOLYSIS
PHOTOSENSITIVITY
POLYMERIZATION
PROTEINS
RADIOISOTOPES
REAGENTS
SENSITIVITY
SUCCINIC ACID
SYNTHESIS
TRACER TECHNIQUES