Guszczynski T, Copeland T D
ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland, 21702-1201, USA.
Anal Biochem. 1998 Jul 1;260(2):212-7. doi: 10.1006/abio.1998.2694.
Affinity capillary electrophoresis was used to detect a shift in mobility when a zinc ion binds to the highly basic nucleocapsid protein (NCp7) of HIV-1. NCp7 contains two Cys-X2- Cys-X4-His-X4-Cys zinc fingers. With constant concentrations of NCp7 as a receptor and various concentrations of zinc as a ligand in the sample buffer and the electrophoresis buffer, we observed changes in electrophoretic mobilities of NCp7 protein when complexes were formed with zinc. Scatchard analysis of the mobility indicates the presence of at least two types of binding sites for zinc. At pH 6.0, one site is shown to bind zinc strongly with a binding constant Kb = 3.25 x 10(5) M-1 and the second site has a Kb = 1.8 x 10(5) M-1. The binding of zinc to the first zinc finger decreased the affinity of zinc for the second zinc finger approximately twofold. The Hill coefficient for this negative cooperativity is 0.9. A series of NCp7 mutants were also examined in the assay to determine their ability to bind zinc. This assay affords a quick method to observe a zinc ion binding to NCp7 and to calculate binding constants.
亲和毛细管电泳用于检测锌离子与HIV-1的高碱性核衣壳蛋白（NCp7）结合时迁移率的变化。NCp7含有两个Cys-X2-Cys-X4-His-X4-Cys锌指结构。在样品缓冲液和电泳缓冲液中，以恒定浓度的NCp7作为受体，不同浓度的锌作为配体，我们观察到当NCp7蛋白与锌形成复合物时其电泳迁移率的变化。对迁移率的Scatchard分析表明锌至少存在两种类型的结合位点。在pH 6.0时，一个位点显示出与锌强烈结合，结合常数Kb = 3.25×10⁵ M⁻¹，第二个位点的Kb = 1.8×10⁵ M⁻¹。锌与第一个锌指结构的结合使锌对第二个锌指结构的亲和力降低了约两倍。这种负协同性的希尔系数为0.9。还在该测定中检测了一系列NCp7突变体，以确定它们结合锌的能力。该测定提供了一种快速方法来观察锌离子与NCp7的结合并计算结合常数。
