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-[125I] 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 125I 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 125I 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