Fassina G, Swaisgood H E, Chaiken I M
J Chromatogr. 1986 Apr 11;376:87-93. doi: 10.1016/s0378-4347(00)80825-7.
To examine analytical high-performance affinity chromatography as a microscale method for characterizing macromolecular interactions, the chromatographic behavior was evaluated of Arg8-vasopressin on bovine neurophysin II covalently immobilized in its monomer form on several new high-flow and pressure-resisting affinity supports. Zonal elution of both tritiated and unlabeled peptide hormone and an extension of theoretical treatment of analytical affinity chromatography allowed determination of equilibrium dissociation constants of hormone binding to immobilized bovine neurophysin II. Microamounts of hormone, ranging from 0.05 to 15 micrograms, were eluted within 20-30 min, with a quantitative recovery of the amount injected. For zones containing more than 5 micrograms, continuous elution monitoring was possible by ultraviolet absorbance, providing greater speed and accuracy in data analysis. The values obtained for the equilibrium dissociation constants were in good agreement with those previously measured in solution. The above hormone-protein evaluation system has led to identification of several pressure-resistant affinity supports, including silica-, agarose- and glass-based matrices, which are appropriate for use with high-performance liquid chromatographic instrumentation for affinity chromatographic analysis of macromolecular interactions.
为了研究分析型高效亲和色谱法作为一种表征大分子相互作用的微量方法，我们评估了精氨酸加压素在以单体形式共价固定在几种新型高流量和耐压亲和支持物上的牛神经垂体素II上的色谱行为。对氚标记和未标记的肽激素进行区带洗脱，并对分析亲和色谱法进行理论扩展，从而能够测定激素与固定化牛神经垂体素II结合的平衡解离常数。0.05至15微克的微量激素在20 - 30分钟内被洗脱，注入量实现了定量回收。对于含有超过5微克的区带，可以通过紫外吸光度进行连续洗脱监测，从而在数据分析中提供更高的速度和准确性。获得的平衡解离常数的值与先前在溶液中测量的值高度一致。上述激素 - 蛋白质评估系统已导致鉴定出几种耐压亲和支持物，包括基于硅胶、琼脂糖和玻璃的基质，这些支持物适用于与高效液相色谱仪器一起用于大分子相互作用的亲和色谱分析。
