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 macro-molecules. 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.
Morag
E
E
Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, Israel.
Bayer
E A
EA
Wilchek
M
M
D001360:Avidin / Q000737:chemistry* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry; D001665:Binding Sites; D001710:Biotin / Q000737:chemistry / Q000378:metabolism*; D002846:Chromatography, Affinity; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011193:Potassium Iodide / Q000494:pharmacology; D012710:Serum Albumin, Bovine / Q000302:isolation & purification; D019809:Streptavidin; D013774:Tetranitromethane / Q000494:pharmacology; D014443:Tyrosine
D001426:Bacterial Proteins; D001360:Avidin; D011193:Potassium Iodide; D012710:Serum Albumin, Bovine; D014443:Tyrosine; D001710:Biotin; D019809:Streptavidin; D013774:Tetranitromethane