Biotinylation of proteins is a powerful tool for investigating biological phenomenon, both in vitro and in vivo. Biotinylating reagents that form covalent bonds with several types of amino acid residues are commercially available. However, most, if not all, of these commercially available biotinylating agents produce biotin–protein bonds that are susceptible to cleavage in human plasma. Here, we describe the use of immunoglobulin G as a model protein for evaluation of biotin–protein bond stability and for the investigation of the mechanism of biotin release. We also describe the synthesis of a biotin–protein bond that is stable in human plasma and a method for evaluation of that stability.
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References
Bogusiewicz, A., Mock, N. I., and Mock, D. M. (2004). Instability of the biotin-protein bond in human plasma. Anal. Biochem. 327, 156–161.
Bogusiewicz, A., Mock, N. I., and Mock, D. M. (2004). Release of biotin from biotinylated proteins occurs enzymatically and nonenzymatically in human plasma. Anal. Biochem. 331, 260–266.
Bogusiewicz, A., Mock, N. I., and Mock, D. M. (2004). A biotin-protein bond with stability in plasma. Anal. Biochem. 337, 98–102.
Mock, D. M. and Malik, M. I. (1992). Distribution of biotin in human plasma: most of the biotin is not bound to protein. Am. J. Clin. Nutr. 56, 427–432.
Mock, D. M. (1997). Determinations of biotin in biological fluids. In Methods in Enzymology (McCormick, D. B., Suttie, J. W. and Wagner, C., eds.), Academic Press, New York, NY. 279, 265–275.
Mock, D. M., Lankford, G. L., and Mock, N. I. (1995). Biotin accounts for only half of the total avidin-binding substances in human serum. J. Nutr. 125, 941–946.
Acknowledgments
This work was supported by PO1 HL 46925 of the National Institute of Heart, Blood, and Lung and by RO1 DK 36823 of the National Institutes of Diabetes, Digestive, and Kidney Diseases.
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© 2008 Humana Press, a part of Springer Science+Business Media, LLC
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Mock, D.M., Bogusiewicz, A. (2008). Biotin–Protein Bond: Instability and Structural Modification to Provide Stability for In Vivo Applications. In: McMahon, R.J. (eds) Avidin-Biotin Interactions. Methods In Molecular Biology™, vol 418. Humana Press. https://doi.org/10.1007/978-1-59745-579-4_17
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DOI: https://doi.org/10.1007/978-1-59745-579-4_17
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