Heterobifunctional cross-linking agents incorporating perfluorinated aryl azides

: New heterobifunctional cross-linking reagents were developed that possess a photoactive tetrafluorinated phenyl azide as the photoactive terminus and a chemically reactive succinimidyl ester as the electrophilic terminus. These reagents, succinimidyl N-(4-azido-2,3,5,6-tetrafluorobenzoyl)tyrosinate (9) and succinimidyl 2-(4-azido-2,3,5,6-tetrafluorophenyl)thiazole-4-carboxylate (15), were designed to possess either an 125I or 35S radiolabel, respectively. In a biochemical study, the latter reagent was coupled to Lys-75 of calmodulin (CaM), and the radioiodinated monoadduct was photochemically cross-linked, in a calcium-dependent manner, to the porcine erythrocyte plasma membrane Ca2+, Mg2(+)-ATPase. Densitometry scans of the gel indicated a reproducible 22% cross-linking of the CaM with one of the Ca2+,Mg2(+)-ATPase bands. Since the purification of the Ca2+,Mg2(+)-ATPase results in micelles having Ca2+,Mg2(+)-ATPase with its CaM binding site oriented both to the inside and outside of the micelle, the amount of Ca2+,Mg2(+)-ATPase available for cross-linking was reduced by approximately half, suggesting that the actual cross-linking efficiency was on the order of 40%.