Morag E, Bayer E A, Wilchek M
Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot, Israel.
Biochem J. 1996 May 15;316 ( Pt 1)(Pt 1):193-9. doi: 10.1042/bj3160193.
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.
维生素生物素与抗生物素蛋白之间的紧密相互作用非常强烈（解离常数Ka约为10¹⁵ M⁻¹），以至于通常足以使蛋白质变性的条件都无法使抗生物素蛋白 - 生物素复合物解离。为了在这两种生物分子之间形成可逆相互作用，我们通过硝化作用修饰了结合位点酪氨酸，从而降低了与生物素脲基形成关键氢键的酚基团的pKa。在相对较低的pH值（4 - 5）下，所得的抗生物素蛋白修饰形式与生物素结合时具有非常高的缔合常数（> 10⁹ M⁻¹）。因此，修饰后的抗生物素蛋白能够支持生物素或生物素化大分子的稳定、长期结合。通过提高介质的pH值或在中性pH下引入过量的生物素，可以使后一种分子分离。这些发现证明了单个氢键对于生物素强结合的重要性。抗生物素蛋白的新衍生物对于需要四个生物素结合位点与生物素之间形成可逆相互作用的应用应该是有用的，从而增加了抗生物素蛋白 -生物素系统在生物技术应用中的多功能性。
