Abstract

A photoreactive N-acetylglucosamine derivative, N-[2-[2-[2-(2-biotinylaminoethoxy)-ethoxy]ethoxy]-4-[3-(trifluoromethyl)-3 H- diazirin-3-yl]benzoyl]-N 4-[2- (acetylamino)-2-deoxy-β- d-glucopyranosyl]- l-aspartamide (BDGA), was synthesized as a carbene-generating biotinylated probe for UDP-galactose: N-acetylglucosamine β-(1 → 4)-galactosyltransferase (GalT). The photoaffinity labeling experiments of bovine GalT with BDGA under various conditions were examined based on the quantitative chemiluminescent detection of the biotinyl residue which was photochemically introduced into the GalT protein. A progressive decrease in the yield of specific photolabeling was observed upon lowering the incubation temperature from 37 °C to 20 °C or 4 °C. The amount of photoincorporation was also decreased when UMP was not included in the incubation mixture. Using a crude protein mixture of recombinant human GalT, a band corresponding to the glutathione S-transferase fusion GalT protein was also specifically visualized. Furthermore, combined use of BDGA photolabeling with an immobilized avidin was found to be effective for the selective retrieval of photolabeled GalT from a reaction mixture containing a large amount of unlabeled GalT protein. The results obtained clearly demonstrate that the covalent biotinylation using the carbene-generating photoaffinity reagent BDGA would be useful for the analysis of acceptor substrate binding sites within the GalT protein.

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