Avidin Assay

The biotin-binding capacity of avidin is determined using the spectrophotometric method of Green (1963). One unit will bind one microgram of d-biotin at pH 8.9.

Reagents

• 0.2 M Ammonium carbonate, pH 8.9
• 0.001 M d-biotin (24.4 mg/100 ml) in ammonium carbonate buffer, pH 6.9

Avidin sample

Dissolve at 0.2 mg/ml in 0.2 M ammonium carbonate buffer, pH 8.9.

Procedure

Adjust spectrophotometer to 233 nm.

Add 3 ml of stock avidin to control and test cuvette. Add 10 ul of d-biotin to test cuvette and record ΔA₂₃₃. Continue adding 10 ul biotin at a time to the test cuvette until no further increase in A₂₃₃ occurs after three successive biotin additions. Plot the number of aliquots of biotin used versus A₂₃₃. Determine the equivalence point by drawing a straight line through the linear absorbance portion of the curve and by drawing a line through the portion where no further increase in A₂₃₃ is observed. The intersection of these lines is the equivalence point. Determine the number of aliquots represented by the equivalence point.

Calculation