Eu(III) luminescence and tryptophan fluorescence spectroscopy as a tool for understanding regioselective hydrolysis of hen egg white lysozyme by metal-substituted Keggin type polyoxometalates

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 5L6←7F0 transition becomes the most intense peak. In the absence of protein, the number of coordinated water molecules to the Eu(III) center 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 5D0→7F2 transition for the Eu-Keggin/HEWL mixture. The calculated association constant amounted to 2.2×102 M-1 for the Eu-Keggin/HEWL complex. Tryptophan fluorescence quenching studies were performed and the quenching constants were calculated to be 9.1×104 M-1, 4×104 M-1 and 4.1×105 M-1for 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.status: publishe