Swaisgood H E, Chaiken I M
Biochemistry. 1986 Jul 15;25(14):4148-55. doi: 10.1021/bi00362a024.
Bovine neurophysin II (BNP II) was covalently immobilized on both nonporous and porous (200-nm pore diameter) glass beads and incorporated in a high-performance liquid chromatograph to evaluate analytical high-performance affinity chromatography as a microscale method for characterizing biomolecular interactions. By extension of the theoretical treatment of analytical affinity chromatography, both the self-association of neurophysin and its binding of the peptide hormone vasopressin were characterized by using a single chromatographic column containing immobilized neurophysin predominantly in the monomer form. Both [3H] [Arg8]vasopressin (AVP) and 125I-BNP II were rapidly eluted (less than 25 min). The relatively symmetrical elution peaks obtained allowed calculation of both equilibrium dissociation constants and kinetic dissociation rate constants. The dissociation constant measured chromatographically for the AVP-immobilized neurophysin complex, KM/L = 11 microM with porous glass beads and 75 microM with nonporous glass (NPG) beads, was in reasonable agreement with those previously obtained by curve fitting of Scatchard plots (16-20 microM) and from binding to [BNP II]Sepharose (50 microM). The values obtained are larger than that for dissociation of AVP from BNP II dimer, by a factor consistent with the intended nature of immobilized BNP II as monomers. Chromatography of BNP II on the [BNP II]NPG gave a dimer dissociation constant of 166 microM, a value in excellent agreement with that derived from equilibrium sedimentation studies (172 microM). In contrast to the agreement of chromatographic equilibrium binding constants with those measured in solution, the dissociation rate, k-3, determined from the variance of the affinity chromatographic elution profile with nonporous beads, was several orders of magnitude smaller than the solution counterpart.(ABSTRACT TRUNCATED AT 250 WORDS)
牛神经垂体素II（BNP II）被共价固定在无孔和多孔（孔径200纳米）玻璃珠上，并整合到高效液相色谱仪中，以评估分析型高效亲和色谱法作为一种表征生物分子相互作用的微尺度方法。通过扩展分析亲和色谱法的理论处理，使用主要以单体形式固定有神经垂体素的单个色谱柱，对神经垂体素的自缔合及其与肽激素加压素的结合进行了表征。[3H][Arg8]加压素（AVP）和125I-BNP II均被快速洗脱（不到25分钟）。所获得的相对对称的洗脱峰使得能够计算平衡解离常数和动力学解离速率常数。通过色谱法测得的AVP-固定化神经垂体素复合物的解离常数，对于多孔玻璃珠，KM/L = 11微摩尔，对于无孔玻璃（NPG）珠为75微摩尔，与先前通过Scatchard图曲线拟合（16 - 20微摩尔）以及与[BNP II]琼脂糖结合获得的值（50微摩尔）合理一致。所获得的值比AVP从BNP II二聚体解离的值大，其倍数与固定化BNP II作为单体的预期性质一致。BNP II在[BNP II]NPG上的色谱分析给出二聚体解离常数为166微摩尔，该值与平衡沉降研究得出的值（172微摩尔）非常一致。与色谱平衡结合常数与溶液中测得的常数一致相反，由无孔珠的亲和色谱洗脱曲线的方差确定的解离速率k - 3比溶液中的对应值小几个数量级。（摘要截断于250字）
