Abstract
A rapid and sensitive spectrophotometric assay was developed for the measurement of biotin-binding sites of immobilized avidin. The method is based on the reaction of avidin with excess biotin followed by assay of the unbound biotin using the HABA (2-[4′-hydroxyazobenzene] benzoic acid) method. Three solids possessing variable amounts of monomeric avidin were examined; viz., succinamidopropyl-controlled-pore glass (CPG-500), crosslinked 6% beaded agarose (Sepharose-CL-6B**), and crosslinked bis-acrylamide/azlactone (3M Emphaze Biosupport Medium AB1. Results indicate that the total biotin-binding sites of monomeric avidin immobilized on CPG-500, Sepharose-CL-6B, and 3M Emphaze are 0.229, 0.093, and 0.218 µmol biotin per mL beads, respectively. Assays for exchangeable biotinbinding sites gave values greater than 90% of the total sites. The spectrophotometric HABA method described is an alternative to assays based on tracers, thus the handling of radioactive material is avoided.
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Green, N. M. and Toms, E. J. (1973),Biochem. J. 133, 687.
Gitlin, G., Bayer, E. A., and Wilchek, M. (1987),Biochem. J. 242, 923.
Henrikson, K. P., Allen, S. H. G., and Maloy, W. L. (1979),Anal. Biochem. 94, 366.
Kohanski, R. A. and Lane, M. D. (1990), inMethods in Enzymology, vol. 184, Wilchek, M. and Bayer, E. A., eds., pp. 194–200, Academic, New York.
Gravel, R. A., Lam, K. F., Mahuran, D., and Kronis, A. (1980),Arch. Biochem. Biophys. 210, 669.
Kohanski, R. A. and Lane, M. D. (1985),J. Biol. Chem. 260, 5014.
Cook, G. M. W. and Buckie, J. W. (1990),in Methods in Enzymology, vol. 184, Wilchek, M. and Bayer, E. A., eds., pp. 304–314, Academic, New York.
Green, N. M. (1970), inMethods in Enzymology, vol. XVIII(A), McCormick, D. B. and Wright, L. D., eds., pp. 418–424, Academic, New York.
Green, N. M. (1965),Biochem. J. 94, 23c.
Janolino, V. G. and Swaisgood, H. E. (1982),Biotechnol. Bioeng. 24, 1069.
DuVal, G., Swaisgood, H. E., and Horton, H. R. (1984),J. Appl. Biochem. 6, 240.
Swaisgood, H. E. and Horton, H. R. (1987),in Methods in Enzymology, vol. 143, Jakoby, W. B. and Griffith, O. W., eds., pp. 504–510, Academic, New York.
Bonde, M., Pontopiddan, H., and Pepper, D. S. (1992),Anal. Biochem. 200, 195.
Pierce (1989), inImmunoPure HABA Reagent Manual, Pierce Chemical Co., Rockford, IL.
Swaisgood, H. E., Horton, H. R., and Mosbach, K. (1976), inMethods in Enzymology, vol. XLIV, Mosbach, K., ed., pp. 504–515, Academic, New York.
Swaisgood, H. E., Janolino, V. G., and Horton, H. R. (1978),Arch. Biochem. Biophys. 191, 259.
DuVal, G., Swaisgood, H. E., and Horton, H. R. (1985),Biochemistry 24, 2067.
Swaisgood, H. E. and Chaiken, I. M. (1986),Biochemistry 25, 4148.
Thresher, W. C. and Swaisgood, H. E. (1990),J. Mol. Recogn. 3, 220.
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Janolino, V.G., Fontecha, J. & Swaisgood, H.E. A spectrophotometric assay for biotinbinding sites of immobilized avidin. Appl Biochem Biotechnol 56, 1–7 (1996). https://doi.org/10.1007/BF02787865
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DOI: https://doi.org/10.1007/BF02787865