Goovaerts Vincent, Stroobants Karen, Absillis Gregory, Parac-Vogt Tatjana N
Department of Chemistry, KU Leuven, Celestijnenlaan 200F, 3001 Leuven, Belgium.
Department of Chemistry, KU Leuven, Celestijnenlaan 200F, 3001 Leuven, Belgium.
J Inorg Biochem. 2015 Sep;150:72-80. doi: 10.1016/j.jinorgbio.2015.03.015. Epub 2015 Apr 1.
The interaction between the lacunary Keggin K7PW11O39, the Eu(III)-substituted Keggin K4EuPW11O39 (Eu-Keggin) and the Ce(IV)-substituted Keggin [Me2NH2]10[Ce(PW11O39)2] (Ce-Keggin) polyoxometalates (POMs), and the proteins hen egg white lysozyme (HEWL) and the structurally homologous α-lactalbumin (α-LA) was studied by steady state and time-resolved Eu(III) luminescence and tryptophan (Trp) fluorescence spectroscopy. The excitation spectrum of Eu-Keggin at lower concentrations ([Eu-Keggin]<100 μM) is dominated by a ligand-to-metal charge transfer band (291 nm). For higher concentrations ([Eu-Keggin]>250 μM) the (5)L6←(7)F0 transition becomes the most intense peak. In the absence of protein, the number of coordinated water molecules to the Eu(III) centre of Eu-Keggin is 4, indicating a 1:1 Eu(III):POM species. In the presence of phosphate buffer this number linearly decreases from 4 to 2 upon increasing phosphate buffer concentration. Upon addition of HEWL, there are no coordinated water molecules, suggesting interaction between Eu-Keggin and the protein surface. In addition, this interaction results in a more than threefold increase of the hypersensitive (5)D0→(7)F2 transition for the Eu-Keggin/HEWL mixture. The calculated association constant amounted to 2.2×10(2) M(-1) for the Eu-Keggin/HEWL complex. Tryptophan fluorescence quenching studies were performed and the quenching constants were calculated to be 9.1×10(4) M(-1), 4×10(4) M(-1) and 4.1×10(5) M(-1) for the lacunary Keggin/HEWL, the Eu-Keggin/HEWL and the Ce-Keggin/HEWL complexes, respectively. The number of bound POM molecules to HEWL was 1.04 for the lacunary Keggin POM, and 1.0 for Eu-Keggin, indicating the formation of a 1:1 POM/HEWL complex. The value of 1.38 for Ce-Keggin might indicate a transition from 1:1 to 1:2 interaction.
通过稳态和时间分辨的铕（III）发光以及色氨酸（Trp）荧光光谱，研究了缺位型Keggin结构的K7PW11O39、铕（III）取代的Keggin结构的K4EuPW11O39（铕-Keggin）和铈（IV）取代的Keggin结构的[Me2NH2]10[Ce(PW11O39)2]（铈-Keggin）多金属氧酸盐（POMs）与蛋清溶菌酶（HEWL）及结构同源的α-乳白蛋白（α-LA）之间的相互作用。较低浓度（[铕-Keggin]<100μM）下铕-Keggin的激发光谱以配体到金属的电荷转移带（291nm）为主。对于较高浓度（[铕-Keggin]>250μM），(5)L6←(7)F0跃迁成为最强峰。在没有蛋白质的情况下，与铕-Keggin的铕（III）中心配位的水分子数为4，表明是1:1的铕（III）:POM物种。在存在磷酸盐缓冲液的情况下，随着磷酸盐缓冲液浓度增加，该数值从4线性降低至2。加入HEWL后，没有配位水分子，这表明铕-Keggin与蛋白质表面存在相互作用。此外，这种相互作用导致铕-Keggin/HEWL混合物的超灵敏(5)D0→(7)F2跃迁增加了三倍多。铕-Keggin/HEWL复合物的计算缔合常数为2.2×10(2) M(-1)。进行了色氨酸荧光猝灭研究，缺位型Keggin/HEWL、铕-Keggin/HEWL和铈-Keggin/HEWL复合物的猝灭常数分别计算为9.1×10(4) M(-1)、4×10(4) M(-1)和4.1×10(5) M(-1)。缺位型Keggin POM与HEWL结合的POM分子数为1.04，铕-Keggin为1.0，表明形成了1:1的POM/HEWL复合物。铈-Keggin的1.38值可能表明从1:1相互作用转变为1:2相互作用。
