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 4 h 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.