AVIDIN. 1. THE USE OF [14C]BIOTIN FOR KINETIC STUDIES AND FOR ASSAY

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Abstract
[2[image]''-Cl4]Biotin has been synthesized from C14O in 70% radiochemical yield. A rapid and simple assay for avidin has been worked out, based on the determination of the excess of [C14] biotin after adsorption of the avidin-biotin complex on carboxymethylcellulose. The method can also be used for the determination of unlabelled biotin. Preliminary kinetic measurements show that the rate constant for avidin-biotin combination (7 X 107 M -1 sec.-1) is of the same order as that for a number of enzyme-substrate-combination reactions. The rate constant for dissociation was very low (9 x 10-8 sec.-l), which accounts for the apparent irreversibility of the avidin inhibition of biotin-containing enzymes. The dissociation constant of the complex calculated from the rate constants was about 10-15 M, corresponding to a free energy change of 20 kcal./mole of biotin bound.