Derivatization and photolysis of a photoaffinity reagent for probing protein and cell surface interactions
The synthesis of the novel, heterobifunctional, cleavable, photoactivable crosslinking reagent, N-(4-(p-azido-m-(/sup 125/I) iodophenylazo)benzoyl)-3-aminopropyl-N'-oxysulfosuccinimide has been described by Denny and Blobel. This reagent is desirable because after photolysis and azo bond cleavage the /sup 125/I is transferred from the reagent to the crosslinked molecule. The authors demonstrate that using the reported synthesis 99% of the desired reagent is destroyed during the chloramine-T iodination step. They report a synthesis revision which produces high yields of the uniodinated (U) reagent. The derivatized reagent may be used in its iodinated (I) or U forms. To study the U reagent, a horseradish peroxidase (HRP) molecule is derivatized with nine reagent molecules. The derivatized HRP has 70% of its original enzymatic activity. After photolysis, 14% of this activity is retained and SDS-PAGE electrophoresis shows a crosslinked complex of HRP molecules. After endocytosis by cells, photolysis attaches the soluble derivatized HRP to membranes allowing them to be traced in the electron microscope. To study the I reagent, an amino-dextran (MW 73-400) molecule is derivatized with three U reagent molecules. The U reagent molecules are then iodinated by the chloramine-T method. With photolysis and cleavage, the /sup 125/I labeled reagent on dextran transfers its label to bovine serum albumin or ovalbumin. The authors conclude this reagent is a versatile probe for study of protein or cell surface topography.
- Research Organization:
- NHLBI, Bethesda, MD
- OSTI ID:
- 6943167
- Report Number(s):
- CONF-8606151-
- Journal Information:
- Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Vol. 45:6; ISSN FEPRA
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
ALBUMINS
BETA DECAY RADIOISOTOPES
CELL CONSTITUENTS
CELL MEMBRANES
CHEMICAL REACTIONS
CLEAVAGE
CROSS-LINKING
CRYSTAL STRUCTURE
DAYS LIVING RADIOISOTOPES
DECOMPOSITION
ELECTRON CAPTURE RADIOISOTOPES
ELECTROPHORESIS
ENZYME ACTIVITY
ENZYMES
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
MEMBRANES
MICROSTRUCTURE
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
OXIDOREDUCTASES
PEROXIDASES
PHOTOCHEMICAL REACTIONS
PHOTOLYSIS
POLYMERIZATION
PROTEINS
RADIOISOTOPES
REAGENTS
TRACER TECHNIQUES