title: Release of biotin from biotinylated proteins occurs enzymatically and nonenzymatically in human plasma.
abstract: Studies in our laboratory and others indicate that biotin is released from biotinylated proteins in vivo and in vitro in human plasma. Using immunoglobulin G (IgG) as the model protein and four different biotinylating reagents, we investigated the mechanism of release. All of the biotin bonds shared an amide link to the carboxyl group of biotin but differed in the chemical links (amide, thioether, and hydrazone) between spacer arm and the various functional groups on IgG. Biotinylated IgG was incubated with phosphate-buffered saline, plasma, or plasma treated to either inactivate enzymes or remove all macromolecules. Released biotin was separated from bound biotin by ultrafiltration and quantitated by avidin-binding assay. As judged by high-performance liquid chromatography, greater than 95% of the released avidin-binding activity was biotin. We infer that the amide bond between the biotin and the spacer arm rather than the bond attaching the spacer to the protein was cleaved. Sodium dodecyl sulfate gel electrophoresis detected no proteolytic degradation of biotinylated IgG. Neither heat inactivation of plasma nor ultrafiltration of plasma to remove macromolecules completely eliminated biotin cleavage. We conclude that cleavage of biotin from protein occurs by both enzymatic and nonenzymatic mechanisms.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.
authors.forename: Anna
authors.initials: A
authors.lastname: Bogusiewicz
authors.forename: Nell I
authors.initials: NI
authors.lastname: Mock
authors.forename: Donald M
authors.initials: DM
authors.lastname: Mock
mesh_terms: D001710:Biotin / Q000737:chemistry*; D001769:Blood; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D011506:Proteins / Q000737:chemistry*
chemical_list: D011506:Proteins; D001710:Biotin