A SPECTROPHOTOMETRIC ASSAY FOR BIOTIN-BINDING SITES OF IMMOBILIZED AVIDIN

A rapid and sensitive spectrophotometric assay was developed for the m easurement of biotin-binding sites of immobilized avidin. The method i s 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 a vidin were examined; viz., succinamido-propyl-controlled-pore glass (C PG-500), crosslinked 6% beaded agarose (Sepharose-CL-6B*), and crossl inked bis-acrylamide/azlactone (3M Emphaze Biosupport Medium AB(1)). R esults indicate that the total biotin-binding sites of monomeric avidi n immobilized on CPG-500, Sepharose-CL-6B, and 3M Emphaze are 0.229, 0 .093, and 0.218 mu mol biotin per mL beads, respectively. Assays for e xchangeable biotin-binding sites gave values greater than 90% of the t otal sites. The spectrophotometric HABA method described is an alterna tive to assays based on tracers, thus the handling of radioactive mate rial is avoided.