title: Instability of the biotin-protein bond in human plasma.
abstract: Labeling proteins with biotin offers an alternative to labeling with radioisotopes for pharmacokinetic studies in humans. However, stability of the biotin-protein bond is a critical tacit assumption. Using release of biotin from immunoglobulin G as the outcome, we individually evaluated stability of the biotin label produced by six biotinylation agents: biotin PEO-amine, 5-(biotinamido)-pentylamine, iodoacetyl-LC-biotin, NHS-LC-biotin, sulfo-NHS-LC-biotin, and biotin-LC-hydrazide. Each of the six biotinylated proteins was incubated at room temperature for 4h in human plasma or in phosphate-buffered saline (control). Free biotin was separated from the biotinylated protein by ultrafiltration and quantitated by avidin-binding assay. For each biotinylation reagent, biotin release was significantly increased by plasma (p < 0.0001 vs control by unpaired t test). Moreover, the hydrazide bond was also unstable in buffer. Biotin remaining on the protein was quantitated directly using capture of europium-streptavidin by the immobilized biotinylated immunoglobulin G. Consistent with biotin release data, streptavidin capture was reduced by plasma to 8% of control. We conclude that all of the biotinylating agents produce biotin-protein bonds that are susceptible to hydrolysis by factors present in human plasma; five of six are stable in buffer.
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: D000818:Animals; D001710:Biotin / Q000737:chemistry*; D019921:Biotinylation / Q000379:methods*; D001798:Blood Proteins / Q000737:chemistry*; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis / Q000737:chemistry; D011485:Protein Binding; D011817:Rabbits
chemical_list: D001798:Blood Proteins; D007074:Immunoglobulin G; D001710:Biotin