Hutchens T W, Yip T T
USDA/ARS Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030.
Anal Biochem. 1990 Nov 15;191(1):160-8. doi: 10.1016/0003-2697(90)90403-v.
The interaction of proteins with immobilized transition-metal ions proceeds via mechanisms influenced by metal type and degree of coordination, variations in mobile phase constituents, and protein surface architecture at or near the metal binding site(s). The contributions each of these variables make toward the affinity of protein surfaces for immobilized metal ions remain empirical. We have used equilibrium binding analyses to evaluate the influence of pH and competitive binding reagents on the apparent equilibrium dissociation constant (Kd) and binding capacity of immobilized Cu(II) and Ni(II) ions for several model proteins of known three-dimensional structure. Linear Scatchard plots suggested that 8/13 of the proteins evaluated interacted with immobilized metal ions via a single class of operational (Kd = 10-700 microM) binding sites. Those proteins with the highest affinities for the immobilized Cu(II) ions (5/13) showed evidence of multiple, non-identical or nonindependent binding sites. The effects of altered metal type, pH, and concentration of competitive affinity reagents (e.g., imidazole, free metal ions) on the apparent Kd and binding capacity varied in magnitude for individual proteins. The presence of free Cu(II) ions did not detectably alter either the affinity or binding capacity of the proteins for immobilized Cu(II) ions. The expected relationship between the relative chromatographic elution sequence and calculated affinity constants was not entirely evident by evaluation under only one set of conditions. Our results demonstrate the utility of nonchromatographic equilibrium binding analyses for the quantitative evaluation of experimental variables affecting the relative affinity and capacity of immobilized metal ions for proteins. This approach affords the opportunity to improve understanding and to vary the contribution of interaction mechanisms involved.
蛋白质与固定化过渡金属离子的相互作用是通过受金属类型和配位程度、流动相成分变化以及金属结合位点处或附近蛋白质表面结构影响的机制进行的。这些变量对蛋白质表面与固定化金属离子亲和力的各自贡献仍基于经验。我们利用平衡结合分析来评估pH值和竞争性结合试剂对固定化Cu(II)和Ni(II)离子与几种已知三维结构的模型蛋白质的表观平衡解离常数（Kd）和结合能力的影响。线性Scatchard图表明，所评估的蛋白质中有8/13通过一类操作（Kd = 10 - 700 microM）结合位点与固定化金属离子相互作用。那些对固定化Cu(II)离子亲和力最高的蛋白质（5/13）显示出多个非相同或非独立结合位点的证据。改变金属类型、pH值和竞争性亲和试剂（如咪唑、游离金属离子）浓度对表观Kd和结合能力的影响因单个蛋白质而异。游离Cu(II)离子的存在并未明显改变蛋白质对固定化Cu(II)离子的亲和力或结合能力。仅在一组条件下进行评估时，相对色谱洗脱顺序与计算出的亲和常数之间的预期关系并不完全明显。我们的结果证明了非色谱平衡结合分析在定量评估影响固定化金属离子与蛋白质相对亲和力和能力的实验变量方面的实用性。这种方法提供了增进理解并改变所涉及相互作用机制贡献的机会。
