First Synthetic Probe for the Detection and Quantification of a Protein with a Potential α,(2→8)Sialyltransferase Activity

• Published in: Bioconjugate chemistry 2004-09, Vol.15 (5), p.1084-1087
• Main Authors: D'Andrea, Gabriele, Lizzi, Anna Rita, Oratore, Arduino
• Format: Article
• Language: English
• Subjects: Biochemistry; Cell Line, Tumor; Chemical synthesis; Data analysis; Enzymes; Humans; Molecular Probes - analysis; Molecular Probes - chemical synthesis; Polysaccharides - analysis; Polysaccharides - chemical synthesis; Proteins; Proteins - analysis; Sialyltransferases - analysis
• ISSN: 1043-1802; 1520-4812
• DOI: 10.1021/bc049970s

There is considerable interest in monitoring α,(2→8)sialyltransferase (ST8) levels; however, there are few specific and sensitive methods to directly detect and quantitate the protein. This paper reports the development of a synthetic probe composed of oxidized colominic acid coupled to biotinyl-l-lysine hydrazide to detect and quantify ST8 with putative “initiase” activity and its use in three solid-phase applications. The detection limit observed for ST8 purified from K562 cells was ∼2 pg by dot-blot analysis. In Western blots the probe bound and specifically recognized a protein band corresponding to ST8. In ELISA a linear dose response was obtained for pure protein in the range of 50−200 pg. Analysis of 3‘-azido-3‘deoxythymidine-treated cells by all three methods showed a reduction in ST8 compared to control cells; treated cells had 73% of control levels by ELISA. This probe will be useful for studies on the expression ST8 and its role in glycoconjugate biosynthesis.