A biotin-protein bond with stability in plasma.
A nonradioactive label for peptide hormones would be useful for pharmacokinetic studies in infants, children, and pregnant women. Because the binding affinity between biotin and avidin is large (Ka=10(15) M(-1)), biotin could also serve as a covalent label for subsequent detection using a variety of avidin conjugates. However, biotin labels produced by most commercially available biotinylating reagents are rapidly cleaved from protein in plasma. We sought to synthesize a stable biotin label for protein. With the use of immunoglobulin G (IgG) as a model protein, biotin was conjugated through a cysteine residue; a carboxylate group was positioned alpha to the biotinamide bond. Stability of the bond in the presence of plasma and buffer control was assessed by release of biotin. Released biotin was separated from biotinylated IgG by ultrafiltration and was quantitated by an avidin-binding assay. In plasma, less than 0.6% of bound biotin was released. This release rate is not significantly different from buffer and is less than 7% of the release rate for IgG biotinylated by N-hydroxysuccinimide-LC-biotin. We conclude that this biotin-protein bond is stable in plasma. We speculate that many uses of avidin-biotin technology could be improved by using this method for protein labeling.
Bogusiewicz
Anna
A
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.
Mock
Nell I
NI
Mock
Donald M
DM
D000328:Adult; D001710:Biotin / Q000031:analogs & derivatives / Q000737:chemistry*; D019921:Biotinylation; D004355:Drug Stability; D006801:Humans; D015335:Molecular Probes / Q000097:blood / Q000138:chemical synthesis*; D011506:Proteins / Q000737:chemistry*
D015335:Molecular Probes; D011506:Proteins; D001710:Biotin