Reversibility of biotin-binding by selective modification of tyrosine in avidin

The tight interaction between the vitamin biotin and the protein avidin is so strong (Ka approximately 10(15) M-1) that conditions which are usually sufficient for protein denaturation fail to dissociate the avidin-biotin complex. In order to form a reversible interaction between the two biomolecules, we have modified the binding-site tyrosine by nitration, thus reducing the PKa of the phenol group which forms a crucial hydrogen bond with the ureido group of biotin. At relatively low pH values (4-5), the resultant modified forms of avidin bind biotin with a very high association constant (> 10(9) M-1). The modified avidins are thus capable of supporting stable, long-term binding of biotin or biotinylated macromolecules. The latter molecules can be detached by increasing the pH of the medium or by introduction of excess levels of biotin at neutral pH. These findings demonstrate the importance of a single hydrogen bond for strong biotin binding. The new derivatives of avidin should be useful for applications whereby a reversible interaction between the four biotin-binding sites and biotin is desired, thus increasing the versatility of the avidin-biotin system for biotechnological application.