Jenuwine E S, Shaner S L
Department of Chemistry, Wayne State University, Detroit, Michigan 48202, USA.
Anal Biochem. 1996 Nov 15;242(2):228-33. doi: 10.1006/abio.1996.0457.
Quantitative zonal DNA affinity chromatography may be used to determine accurate equilibrium constants for the binding of proteins nonspecifically to DNA. Zonal quantitative affinity chromatography has not previously been applied to the determination of binding constants of proteins to DNA, although its use is quite commonplace for determination of affinity constants for protein-protein or protein-small ligand interactions. Equilibrium constants were measured for the nonspecific binding of bovine pancreatic ribonuclease A and Escherichia coli lac repressor to double-stranded DNA immobilized on cellulose. The equilibrium constants determined agree with literature values evaluated using other techniques. The experimental advantages of the zonal technique, when it can be applied, are that collection of data is fast and data analysis is simple. Detection of the protein elution profile by absorbance at 220 nm with an in-line detector can provide adequate sensitivity when binding constants are in the range 10(2)-10(4) M-1.
定量区域DNA亲和色谱法可用于确定蛋白质与DNA非特异性结合的准确平衡常数。区域定量亲和色谱法此前尚未应用于蛋白质与DNA结合常数的测定，尽管其在测定蛋白质-蛋白质或蛋白质-小分子配体相互作用的亲和常数方面应用十分普遍。我们测定了牛胰核糖核酸酶A和大肠杆菌乳糖阻遏蛋白与固定在纤维素上的双链DNA非特异性结合的平衡常数。所测定的平衡常数与使用其他技术评估的文献值相符。当适用时，区域技术的实验优势在于数据收集快速且数据分析简单。当结合常数在10² - 10⁴ M⁻¹范围内时，使用在线检测器通过220 nm处的吸光度检测蛋白质洗脱曲线可提供足够的灵敏度。
