Original Articles

Applied Biochemistry and Biotechnology

, Volume 56, Issue 1, pp 1-7

First online:

A spectrophotometric assay for biotinbinding sites of immobilized avidin

  • Violeta G. JanolinoAffiliated withDepartment of Food Science, North Carolina State University
  • , Javier FontechaAffiliated withDepartment of Food Science, North Carolina State University
  • , Harold E. SwaisgoodAffiliated withDepartment of Food Science, North Carolina State University

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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.

Index Entries

Spectrophotometric assays biotin-binding sites avidin, immobilized