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A novel photoactivatable crossâlinker for the functionallyâdirected regionâspecific fluorescent labeling of proteins
- THEVENIN, Benard J.âM., SHAHROKH, Zahra, WILLIARD, Renee L., FUJIMOTO, Edward K., KANG, JawâJou, IKEMOTO, Noriaki, SHOHET, Stephen B.
- European journal of biochemistry 1992 v.206 no.2 pp. 471-477
- binding sites, coumarin, cross-linking reagents, crosslinking, fluorescence, fluorescent labeling, photolysis, polyacrylamide gel electrophoresis, proteins, trypsin, trypsin inhibitors
- A cleavable crossâlinking reagent, sulfosuccinimidylâ2(7âazidoâ4âmethycoumarinâ3âacetamido)âethylâ1,3â²âdithiopropionate (SAED), was synthesized for the selective transfer of a coumarin fluorophore from a âdonorâ protein to a position near the binding site of an interacting âtargetâ protein. SAED contains a terminal Nâsulfoscuccinimidyl ester for conjugation to the donor, a terminal photoactivatable azidoâcoumarin species for crossâlinking with the interacting target, and a central disculfide spacer for the release of the labeled target after cleavage. To evaluate the effectiveness of this labeling reagent, soynean trypsin inhibitor (STI) was derivatized (â¼ 0.5 mol/mol) with SAED and then photolyzed in the presence of trypsin. A single fluorescent crossâlinked species (6â7 mol% of total STI) was observed by SDS/PAGE and, after refuctive cleavage, was shown to be a 1: 1 STIâtrypsin complex. This complex was not detected without photolysis or with an inactivated crossâlinker. Importantly, complex formation was inhibited by an excess of unmodified STI and prevented by substitution of a nonâinteracting protein for trypsin. Cleavage of the crossâlinked complex revealed that the trypsin, but not the STI, was fluorescent; the uncomplexed trypsin fraction remained unlabeled. These results demonstrated the specificty of the labeling of trypsin by fluorescentâtransfer crossâlinking with SAED. An efficiency of about 15% for this crossâlinking mediated labeling of trypsin was calculated. The short crossâlinking span of SAED (â¤1.8nm) strictly limited the labeling to the vicinity of the contact region of trypsin with STI. thus, this novel crossâlinker permits the regionâspecific targeting of a fluorophore near a functionally important binding site.