Applied Biochemistry and Biotechnology

, Volume 56, Issue 1, pp 1–7 | Cite as

A spectrophotometric assay for biotinbinding sites of immobilized avidin

  • Violeta G. Janolino
  • Javier Fontecha
  • Harold E. Swaisgood
Original Articles


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.

Index Entries

Spectrophotometric assays biotin-binding sites avidin, immobilized 


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Copyright information

© Humana Press Inc. 1996

Authors and Affiliations

  • Violeta G. Janolino
    • 1
  • Javier Fontecha
    • 1
  • Harold E. Swaisgood
    • 1
  1. 1.Department of Food ScienceNorth Carolina State UniversityRaleigh

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