title: Photoaffinity labeling of sialidase with a biotin-conjugated phenylaminodiazirine derivative of 2,3-didehydro-2-deoxy-N-acetylneuraminic acid.
abstract: A biotin-conjugated photoactivatable phenylaminodiazirine derivative of 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (DANA) was synthesized to identify sialidase. The free carboxylic group and N-acetyl substituent of sialic acid, which are important for recognition and enzymatic activity of sialidase, were conserved by the photolabeling compound as confirmed using analytical methods. The synthesized compound and DANA competitively inhibited starfish sialidase with a Ki value of 7.6 microM and 4.6 microM, respectively. Photo incorporation of the labeling compound to sialidase increased with irradiation time; 90% photo incorporation was achieved with more than 10-min irradiation, and labeling was completely inhibited by the addition of a competitive inhibitor. Starfish sialidase purified using high-performance gel filtration chromatography was subjected to photoaffinity labeling. A 50-kDa band was revealed to contain the sialidase active site by the photolabeling compound, and labeling was completely hindered in presence of the competitive inhibitor. Labeling specificity was ensured by the addition of the heat-deactivated standard protein chymotrypsinogen A to the reaction mixture.
authors.affiliation: Department of Health Chemistry, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata 956-8603, Japan.
authors.forename: Ramaswamy
authors.initials: R
authors.lastname: Kannappan
authors.forename: Masayuki
authors.initials: M
authors.lastname: Ando
authors.forename: Kimio
authors.initials: K
authors.lastname: Furuhata
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Uda
mesh_terms: D000818:Animals; D001710:Biotin / Q000737:chemistry*; D003978:Diazomethane / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D013058:Mass Spectrometry; D019158:N-Acetylneuraminic Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D009439:Neuraminidase / Q000032:analysis*; D010053:Ovary / Q000201:enzymology; D019920:Photoaffinity Labels / Q000138:chemical synthesis / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared; D013215:Starfish / Q000201:enzymology
chemical_list: D019920:Photoaffinity Labels; C036137:2-deoxy-2,3-dehydro-N-acetylneuraminic acid; D003978:Diazomethane; D001710:Biotin; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid