A 125I-radiolabel transfer crosslinking reagent with a novel cleavable group

A new chemical crosslinking reagent, 1-[N-(2-hydroxy-5-azidobenzoyl) -2-aminoethyl]-4-(N-hydroxysuccinimidyl)-succinate, or HAHS, has been developed. It is synthesized in three steps and stored as an unlabeled precursor, and then iodinated immediately before use. The reagent has a succinimide ester at one end so that it can be covalently attached to a purified protein, and a radioiodinated phenyl azide group at the other end, so that upon photolysis it can form crosslinks to nearby molecules. The 16-A connecting region contains an ester group which is very stable at neutral pH before photolysis, but which hydrolyzes in about 1 min in base, and hydrolyzes spontaneously after photolysis. Thus, photolysis and cleavage of the ester result in transfer of the radiolabel from the initial protein to its neighbors. When 125I-HAHS-protein A was incubated with IgG, photolyzed, and cleaved, 27% of the label was transferred to the IgG heavy chain. This transfer was abolished by an excess of unlabeled protein A, and was quenched by low concentrations of DTT. Much lower amounts of label were transferred to noninteracting proteins. When 125I-HAHS-spectrin was bound to spectrin-depleted red blood cell membranes, photolyzed, and cleaved, label was transferred only to ankyrin and to band 3. This transfer was blocked by excess unlabeled spectrin and was greatly diminished by conditions which prevent binding of spectrin.