title: Characterization of morphine-glucose-6-phosphate dehydrogenase conjugates by mass spectrometry.
abstract: A key characteristic of the analyte-reporter enzyme conjugate used in the enzyme-multiplied immunoassay technique (EMIT) is the inhibition of the conjugate enzyme upon anti-analyte antibody binding. To improve our understanding of the antibody-induced inhibition mechanism, we characterized morphine-glucose-6-phosphate dehydrogenase (G6PDH) conjugates as model EMIT analyte-reporter enzyme conjugates. Morphine-G6PDH conjugates were prepared by acylating predominantly the primary amines on G6PDH with morphine 3-glucuronide NHS ester molecules. In this study, morphine-G6PDH conjugates were characterized using a combination of methods, including tryptic digestion, immunoprecipitation, matrix-assisted laser desorption ionization mass spectrometry, and electrospray ionization tandem mass spectrometry. Twenty-six conjugation sites were identified. The identified sites all were found to be primary amines. The degree of conjugation was determined to be less than the number of conjugation sites, suggesting heterogeneity within the morphine-G6PDH conjugate population. Two catalytically important residues in the active site (K22 and K183) were among the identified conjugation sites, explaining at least partially the cause of loss of activity due to the coupling reaction.
authors.affiliation: Department of Chemical and Biomolecular Engineering, David Geffen School of Medicine at UCLA, University of California, Los Angeles, California 90095-1592, United States.
authors.forename: May L
authors.initials: ML
authors.lastname: Chiu
authors.forename: A Jimmy
authors.initials: AJ
authors.lastname: Ytterberg
authors.forename: Rachel R
authors.initials: RR
authors.lastname: Ogorzalek Loo
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Loo
authors.forename: Harold G
authors.initials: HG
authors.lastname: Monbouquette
mesh_terms: D000588:Amines / Q000737:chemistry; D000906:Antibodies; D001665:Binding Sites; D020134:Catalytic Domain; D004791:Enzyme Inhibitors; D017248:Enzyme Multiplied Immunoassay Technique; D005954:Glucosephosphate Dehydrogenase; D013058:Mass Spectrometry / Q000379:methods*; D009020:Morphine
chemical_list: D000588:Amines; D000906:Antibodies; D004791:Enzyme Inhibitors; D009020:Morphine; D005954:Glucosephosphate Dehydrogenase