Since the importance of the high affinity between avidin and biotin, K_d = 3 × 10⁻¹⁶ M, gained universal recognition, numerous chemical, biological and medical avidin-biotin based applications have been developed. However, in some cases the high affinity may be a disadvantage, as this interaction is irreversible under physiological conditions. The dye, 4′-hydroxyazobenzene-2-carboxylic acid (HABA), binds avidin, at the biotin binding site, as determined by X-ray, at a much lower affinity constant, K_d = 6 × 10⁻⁶ M. We prepared a HABA affinity column (amber colored). Avidin bound to the column at a pH between 4 and 8.5, causing a change of color to red, and it could be eluted at mild conditions with buffers containing biotin, HABA, 1.5 M potassium chloride or a pH lower than 4.0 or higher than 8.5. Avidin eluted with HABA, created a red avidin-HABA complex, which was visualized. HABA free avidin was obtained by dialysis, which was followed by the loss of red coloration. The novel and easy to use HABA-affinity column was employed in our lab to prepare pure, fully glycosylated avidin from egg white. Most importantly, it may serve as an ideal tool for educational purposes, illuminating concepts of molecular recognition, reversible molecular binding, structure-based molecular design and solid phase chemical synthesis, as it is a reliable and visible reagent.

由于抗生物素蛋白和生物素之间的高亲和力的重要性（K _d  = 3×10 ^-16  M）已获得普遍认可，因此已开发出许多基于化学，生物和医学的抗生物素蛋白-生物素的应用。但是，在某些情况下，高亲和力可能是不利的，因为这种相互作用在生理条件下是不可逆的。染料4'-羟基偶氮苯-2-羧酸（HABA）在X射线测定的生物素结合位点以高得多的亲和常数K _d  = 6×10 ^-6结合抗生物素蛋白。 M.我们准备了一个HABA亲和柱（琥珀色）。抗生物素蛋白在4至8.5的pH值下与色谱柱结合，导致颜色变为红色，可以在温和条件下用含有生物素，HABA，1.5 M氯化钾或pH值低于4.0或高于8.5的缓冲液洗脱。用HABA洗脱的抗生物素蛋白产生了红色的抗生物素蛋白-HABA复合物，将其可视化。通过透析获得不含HABA的抗生物素蛋白，随后损失红色。我们实验室采用新颖且易于使用的HABA亲和柱从蛋清中制备纯的，完全糖基化的抗生物素蛋白。最重要的是，它是一种可靠的可见试剂，可作为教学目的的理想工具，阐明了分子识别，可逆分子结合，基于结构的分子设计和固相化学合成的概念。