Ma W, Hwang K J, Lee V H
Department of Pharmaceutical Sciences, University of Southern California, School of Pharmacy, Los Angeles 90033.
Pharm Res. 1993 Feb;10(2):204-7. doi: 10.1023/a:1018974424624.
A terbium-dipicolinic acid (Tb-DPA) fluorescence quenching method for estimating free chelating groups conjugated to protein molecules was developed. This method was based on competitive displacement of DPA from binding to terbium by stronger chelating groups such as diethylenetriaminepentaacetic acid (DTPA), EDTA, nitrilotriacetic acid (NTA), DTPA-conjugated bovine serum albumin (BSA-DTPA), or DTPA-conjugated immunoglobulin G (IgG-DTPA), resulting in a significant reduction in terbium fluorescence. The chelating ability of the tested reagent, from high to low, was in the following order: BSA-DTPA > DTPA > IgG-DTPA > EDTA, NTA. At low terbium concentrations, the reduction was linear for DTPA. This fluorescence quenching method was not only rapid, simple, and as accurate as conventional radiosotopic or chromatographic methods, but also sensitive and reproductible. The detection limit was 10 nM for DTPA. The interrun coefficient of variation was at most 8%. The advantage of this method over other indirect methods is that it reveals the actual chelating ability of the tested macromolecule, unencumbered by complicating factors such as trace metal contamination and dimer/polymer formation during conjugation.
开发了一种用于估算与蛋白质分子共轭的游离螯合基团的铽-二吡啶甲酸（Tb-DPA）荧光猝灭法。该方法基于更强的螯合基团（如二乙烯三胺五乙酸（DTPA）、乙二胺四乙酸（EDTA）、次氮基三乙酸（NTA）、DTPA共轭牛血清白蛋白（BSA-DTPA）或DTPA共轭免疫球蛋白G（IgG-DTPA））从与铽的结合中竞争性取代二吡啶甲酸，导致铽荧光显著降低。受试试剂的螯合能力从高到低依次为：BSA-DTPA>DTPA>IgG-DTPA>EDTA、NTA。在低铽浓度下，DTPA的荧光降低呈线性。这种荧光猝灭法不仅快速、简单，且与传统放射性同位素法或色谱法一样准确，而且灵敏且可重复。DTPA的检测限为10 nM。批间变异系数最高为8%。该方法相对于其他间接方法的优势在于，它揭示了受试大分子的实际螯合能力，不受诸如痕量金属污染以及共轭过程中形成二聚体/聚合物等复杂因素的影响。
