Conformational Changes of DNA by Photoirradiation of DNA-Bis(ZnII-Cyclen)-Azobenzene Complex

Bis(Zn^II-cyclen)-azobenzene derivative, which has two Zn^II-macrocyclic tetraamine complexes connected through azobenzene spacer, has been synthesized as a cross-linking agent for double stranded DNA in aqueous solution. The Zn^II-cyclen derivative selectively binds to A-T base pairs producing complexes between the Zn^II-cyclen moiety and the imide-deprotonated thymine with breaking A-T base pairs. The azobenzene spacer undergoes cis/trans photoisomerization in the complex between the Zn^II-cyclen derivative and the DNA duplex. The conformation of the DNA remarkably changed by photoisomerization of the azobenzene linker, when the Zn^II-cyclen derivative binds to the DNA duplex with an interstrand cross-linking manner.     

Effect of Eu3+ on characterization of photosystem II particles from spinach by spectroscopy

Photosystem II (PSII) particles were purified from Eu³⁺-treated spinach and studied by spectroscopy. The results showed that electron transport rate of PS II was accelerated by Eu³⁺ treating, that violet shift of the PSII Soret band or Q-band was 6 nm or 2 nm for the ultraviolet-visible (UV-Vis) spectrum, that the violet shift of the PSII fluorescence emission peak was 9 nm for fluorescence emission spectrum, that the PSII Signal II’s of low-temperature electron paramagnetic resonance (EPR) spectrum was intensified under light, and that the PSII CD spectrum was similar to that of control. It is suggested that Eu³⁺ might bind to the PSII reaction center complex and enhance the electron transport rate of PSII CD; however, Eu³⁺ treatment does not change the configuration of the PSII reaction center complex.     

Spectrofluorimetric determination of bilirubin in serum samples

A new spectrofluorimetric method was developed for determination of trace amount of bilirubin. Using oxytetracycline–Eu³⁺ as a fluorescent probe, in the buffer solution of pH = 7.3, bilirubin can reduce remarkably the fluorescence intensity of the oxytetracycline–Eu³⁺ complex at λ = 612 nm and the reduced fluorescence intensity was in proportion to the concentration of bilirubin. Optimum conditions for the determination of bilirubin were also investigated. The linear range and limit of detection for the determination of bilirubin were 5.0 × 10^?7, 3.0 × 10^?5 and 7.7 × 10^?8 mol L^?1, respectively. This method is simple, practical and relatively free of interference from coexisting substances and can be successfully applied to assess bilirubin in serum samples and compared with the modified Jendrassik–Grof method in clinical analysis. The quenching mechanism of the fluorescence intensity in the system is also discussed. Copyright © 2010 John Wiley & Sons, Ltd.     

155Eu3+ as a probe of metal ion and cationic drug binding sites on native and heat-denatured DNA

A flow-dialysis apparatus suitable for the study of high affinity metal ion binding sites in macromolecules has been utilized to study ¹⁵⁵Eu³⁺ exchange processes, as a function of pH, in both ‘native’ and ‘heat-denatured’ DNA. ‘Free exchange’ of ¹⁵⁵Eu³⁺ was found to occur at a significantly faster rate at pH = 7.0 than at pH = 6.0 for both forms of DNA; while non-radioactive Eu³⁺-induced ‘displacement’ of bound ¹⁵⁵Eu³⁺ occurred at a significantly faster rate at pH = 6.0 than at pH = 7.0 for both species of DNA. These results are consistent with a greater ‘entropic’ driving force for metal ion:DNA complexation at the lower pH value. The effect of ethidium bromide on ¹⁵⁵Eu³⁺ exchange was also examined as a function of pH. The intercalating agent was found to accelerate ¹⁵⁵Eu³⁺ displacement at pH = 6.0 and decelerate displacement at pH = 7.0. All three sets of experiments (i.e., free- exchange of bound ¹⁵⁵Eu³⁺, Eu³⁺-induced displacement of bound ¹⁵⁵Eu³⁺ and ethidium ion-induced displacement of bound ¹⁵⁵Eu³⁺) indicate that the ¹⁵⁵Eu³⁺ ion can serve as a useful probe of metal ion and drug binding sites in nucleic acid polymers, and constitutes a particularly sensitive probe at pH = 6.0.     

Research of the entry of rare earth elements Eu3+ and La3+ into plant cell

Whether rare earth elements can enter into plant cells remains controversial. This article discusses the ultracellular structural localization of lanthanum (La³⁺) and europium (Eu³⁺) in the intact plant cells fed by rare earth elements Eu³⁺ and La³⁺. Eu-TTA fluorescence analysis of the plasmalemma, cytoplast, and mitochondria showed that Eu³⁺ fluorescence intensities in such structures significantly increased. Eu³⁺ can directly enter or be carried by the artificial ion carrier A23187 into plant cells through the calcium ion (Ca²⁺) channel and then partially resume the synthesis of amaranthin in the Amaranthus caudatus growing in the dark. Locations of rare earth elements La³⁺ and Eu³⁺ in all kinds of components of cytoplasmatic organelles were determined with transmission electron microscope, scanning electron microscope, and energy-dispersive X-ray microanalysis. The results of energy-dispersive X-ray microanalysis indicated that Eu³⁺ and La³⁺ can be absorbed into plant cells and bind to the membranes of protoplasm, chloroplast, mitochondrion, cytoplast, and karyon. These results provide experimental evidence that rare earth elements can be absorbed into plant cells, which would be the basis for interpreting physiological and biochemical effects of rare earth elements on plant cells.     

Zinc(II)–cyclen polyacrylamide gel electrophoresis for detection of mutations in short Ade/Thy-rich DNA fragments

We describe an improved gel-based method with an additive Zn²⁺-cyclen complex (cyclen, 1,4,7,10-tetraazacyclododecane), Zn²⁺-cyclen–PAGE, for mutation detection in DNA fragments by PCR that contain more than 65% Ade/Thy bases and fewer than 100 base pairs (bp). Existing techniques have a problem in analyzing such short Ade/Thy-rich fragments because the duplexes are disrupted and are not detectable due to binding of Zn²⁺-cyclen to Thy bases. In this strategy using a PCR primer with a Gua/Cyt-lined sequence attached at its 5′-end, we successfully detected a mutation in an 86-bp Ade/Thy-rich region of the BRCA1 gene from formalin-fixed paraffin-embedded breast cancer-tissue sections.     

One-Step Preparation and Fluorescence Enhancement Effect in SiO2/Eu2+ Doped with Ag Nanowires

Eu²⁺ single-doped SiO₂ (SiO₂/Eu²⁺) and Eu²⁺, Ag nanowires co-doped SiO₂ (SiO₂/Eu²⁺–Ag) luminescent nanomaterials were prepared by an efficient one-step sol–gel method. Their microstructure and optical properties were characterized, and the fluorescence enhancement of Eu²⁺ by Ag nanowires was investigated. The experimental results indicate that the average diameter of Ag nanowires doped is 12.5 nm, and the length–diameter ratio is 30. The Ag nanowires cannot only enhance the light absorption of SiO₂/Eu²⁺ in the range of 230–350 nm, but also reduce the fluorescence lifetime of Eu²⁺. More importantly, the emission intensity is enhanced after doping Ag nanowires, and the red shift phenomenon of the emission spectrum is observed, red shift occurs between 10 and 56 nm. The highest fluorescence intensity is accessed under the Ag doping concentration of 0.10 %. Additionally, the emission of SiO₂/Eu²⁺ with 0.10 % of Ag doping at 456 nm is 16 times stronger than that of pure SiO₂/Eu²⁺. The present results indicate that the fluorescence enhancement is attributed to the local field enhancement and the increased radiative decay rates induced by Ag nanowires.     

Spectral investigations on Eu3+,Sm3+‐doped and Sm3+/Eu3+ co‐doped potassium‐fluoro‐phosphate glass emitting intense orange‐red for lighting applications

Potassium fluoro‐phosphate (KFP) glass singly doped with different concentrations of europium (Eu³⁺) or samarium (Sm³⁺) or co‐doped (Sm³⁺/Eu³⁺) was prepared, and their luminescence spectra were investigated. The phase composition of the product was verified by X‐ray diffraction analysis. Optical transition properties of Eu³⁺ in the studied potassium phosphate glass were evaluated in the framework of the Judd–Ofelt theory. The radiative transition rates (A_R), fluorescence branching ratios (β), stimulated emission cross‐sections (σ_e) and lifetimes (τ_exp) for certain transitions or levels were evaluated. Red emission of Eu³⁺ was exhibited mainly by the ⁵D₀→⁷F₂ transition located at 612 nm. Concentration quenching and energy transfer were observed from fluorescence spectra and decay curves, respectively. It was found that the lifetimes of the ⁵D₀ level increased with increase in concentration and then decreased. By co‐doping with Sm³⁺, energy transfer from Sm³⁺ to Eu³⁺ occurred and contributed to the enhancement in emission intensity. Intense orange‐red light emission was obtained upon sensitizing with Sm³⁺ in KFP glass. This approach shows significant promise for use in reddish‐orange lighting applications. The optimized properties of the Sm³⁺/Eu³⁺ co‐doped potassium phosphate glass might be promising for optical materials.     

Rapid determination of gatifloxacin in biological samples and pharmaceutical products using europium‐sensitized fluorescence spectrophotometry

A europium‐sensitized fluorescence spectrophotometry method using an anionic surfactant, sodium dodecyl benzene sulphonate (SDBS), was developed for the determination of gatifloxacin (GFLX). The GFLX–Eu³⁺–SDBS system was studied and it was found that SDBS significantly enhanced the fluorescence intensity of the GFLX–Eu³⁺ complex (about 25‐fold). The optimal experimental conditions were determined as follows: excitation and emission wavelengths of 338 and 617 nm, pH 7.5, 3.0 × 10^–6 mol/L europium(III), and 5.0 × 10^–5 mol/L SDBS. The enhanced fluorescence intensity of the system (ΔI_f) showed a good linear relationship with the concentration of GFLX over the range 1.0 × 10^–8–8.0 × 10^–7 mol/L with a correlation coefficient of 0.9990. The detection limit (S:N = 3) was determined as 1.0 × 10^–9 mol/L. This method has been successfully applied for the determination of GFLX in pharmaceuticals and human urine/serum samples. Compared with most other methods reported, the rapid and simple procedure proposed here offered higher sensitivity, wider linear range and good stability. The luminescence mechanism of the system is also discussed in detail. Copyright © 2008 John Wiley & Sons, Ltd.     

Interaction of Eu3+ with yeast alcohol dehydrogenase

The activity of yeast alcohol dehydrogenase is markedly enhanced by Eu³⁺ ions. At pH 7.0 two binding constants for Eu³⁺, 1.0 × 10^–2 and 2.0 × 10^–3 M, were obtained using a Scatchard plot. The presence of Zn²⁺ ions restricts the Eu³⁺-induced increase in the activity of yeast alcohol dehydrogenase. Studies on the tryptophan fluorescence of the enzyme in the absence and presence of Eu³⁺ or Zn²⁺ ions showed that Eu³⁺ affects tertiary or quaternary structures, which is consistent with its activation of the enzyme. The presence of Zn²⁺ reverses the conformational changes caused by Eu³⁺. Comparison of the effects of Eu³⁺ with Zn²⁺ for apo-yeast alcohol dehydrogenase indicates that their binding sites on the protein are different.     

Tunable luminescence properties and energy transfer in LaAl11O18:Eu,Tb phosphor

Eu²⁺ and Tb³⁺ singly doped and co‐doped LaAl₁₁O₁₈ phosphors were prepared by a combustion method using urea as a fuel. The phase structure and photoluminescence (PL) properties of the prepared phosphors were characterized by powder X‐ray diffraction (XRD), scanning electron microscopy (SEM), and photoluminescence excitation and emission spectra. When the content of Eu²⁺ was fixed at 0.01, the emission chromaticity coordinates could be adjusted from blue to green region by tuning the contents of Tb³⁺ ions from 0.01 to 0.03 through an energy transfer (ET) process. The fluorescence data collected from the samples with different contents of Tb³⁺ into LaAl₁₁O₁₈: Eu, show the enhanced green emission at 545 nm associated with ⁵D₄ –⁷F₅ transitions of Tb³⁺. The enhancement was attributed to ET from Eu²⁺ to Tb³⁺, and therefore Eu²⁺ ion acts as a sensitizer (an energy donor) while Tb³⁺ ion as an activator. The ET from Eu²⁺ to Tb³⁺ is performed through dipole–dipole interaction. The ET efficiency and critical distance were also calculated. The present Eu²⁺–Tb³⁺ co‐doped LaAl₁₁O₁₈ phosphor will have potential application for UV convertible white light‐emitting diodes. Copyright © 2015 John Wiley & Sons, Ltd.     

Synthesis, crystalline structure and photoluminescence investigations of the new trivalent rare earth complexes (Sm, Eu and Tb) containing 2-thiophenecarboxylate as sensitizer

New complexes with the general formula [RE(TPC)₃ · (H₂O)₂], where RE=Eu³⁺, Sm³⁺, Gd³⁺, Tb³⁺ and TPC=2-thiophenecarboxylate, have been prepared and investigated by photoluminescence spectroscopy. These compounds were characterized by complexometric titration, elemental analyses and infrared spectroscopy. The X-ray crystal structure has been determined for the [Eu(TPC)₃ · (H₂O)₂] compound, indicating that this complex is in dimeric form bridged by two carboxylate ions with monoclinic crystal system and space group P2₁/n. The coordination polyhedron can be described as a distorted square antiprism, where six oxygen atoms belong to the TPC ligand and two oxygen atoms belong to the water molecules, with site symmetry close to C_2v. The theoretical value of the intensity parameter , which is in agreement with the experimental one, indicates that the Eu³⁺ ion is in a highly polarizable chemical environment. Based on the luminescence spectra, the energy transfer from the ligand triplet state (T) of TPC to the excited levels of the Eu³⁺ ion is discussed. The emission quantum efficiency of the ⁵D₀ emitting level of the Eu³⁺ ion was also determined. In the case of the Tb³⁺ ion, the photoluminescence data show the high emission intensity of the characteristic transitions ⁵D₄ → ⁷F_J (J=0-6), indicating that the TPC ligand is a good sensitizer. It is also noticed that the complexes with the Eu³⁺ and Tb³⁺ ions are more luminescent than the complex with the Sm³⁺ ion.     

Magnetic and luminescence characteristics of dinuclear complexes of lanthanides and a phenolic schiff base macrocyclic ligand

The magnetic and luminescence characteristics of trimorphic homodinuclear macrocyclic complexes of lanthanides and a 2:2 phenolate Schiff's base L, derived from 2,6-diformyl-p-cresol and triethylenetetramine were determined. The complexes of Pr³⁺ exhibit non-Curie-Weiss temperature dependent magnetic susceptibilities for which satisfactory fits to an axial relationship depends on crystal field splitting and a weak binuclear Pr³⁺Pr³⁺ antiferromagnetic interaction. The exchange interaction parameters are zJ′ = −2.2, −4.4 and −7.0 cm ⁻¹ for ‘off-white’ Pr₂L(NO₃)₄·2H₂O, ‘yellow’ Pr₂L(NO₃)₄, and ‘orange’ Pr₂L(NO₃)₂(OH)₂, respectively. In contrast, magnetic susceptibilities of the Ln₂L(NO₃)₃(OH) complexes (Ln = Dy, Ho) follow Curie-Weiss behavior over the entire temperature range (6 K to 300 K). The complexes of closed shell ions La³⁺, Lu³⁺, Y³⁺ and those of the half filled shell ion Gd³⁺ exhibit a strong ligand fluorescence in the 450 nm to 650 nm range with decay times at 77 K of 5–8 ns for Ln≠Gd or 2–4 ns for Ln = Gd. The complexes of Gd³⁺ also exhibit a phosphorescence at 600 nm (decay time ∼ 200 μs). The complexes containing Ce³⁺, Eu³⁺, Tb³⁺ and Er³⁺ show very weak ligand luminescence indicative of effective quenching processes. Sensitized emission from the lanthanide ion is observed only with the Eu³⁺ complexes (⁵D_o → ⁷F_j transitions). The emission lifetimes are on the order of 250 μs in the pure Eu³⁺ complexes. The emission decay curves from dilute samples of Eu³⁺ in ‘off-white’ La₂L(NO₃)_4nH₂O show a noticeable rise time as well as a biphasic decay (fast component ∼ 400 μs; slow component ∼ 2500 μs). The luminescing states of L and Eu³⁺ have a common excitation spectrum which is similar to the electronic absorption spectrum of L indicating that ligand-to-metal ion energy transfer processes are dominant. Overall the result if this study suggest that the spectral properties of the complexes are determined by the coordination mode of the lanthanide ions to the Schiff base portion of macrocyclic ligand.     

A novel Nudix hydrolase for oxidized purine nucleoside triphosphates encoded by ORFYLR151c (PCD1 gene) in Saccharomyces cerevisiae

A search for candidates for a functional homologue of Escherichia coli MutT in yeast Saccharomyces cerevisiae was made in the NCBI-BLAST database using the Nudix box, a short amino acid sequence conserved among E.coli MutT, Pseudomonoas vulgaris MutT, and human, rat and mouse MTH1. Among five candidates, we focused on the open reading frame YLR151c, because it had a region with ~76% similarity to the N-terminal half of MutT including the Nudix box. We thus evaluated the ability of YLR151c as a functional homologue of E.coli MutT in S.cerevisiae. Expression of YLR151c was able to suppress the transversion from A:T to C:G caused by misincorporation of the oxidized nucleotide 8-oxo-dGTP in the E.coli mutT-deficient strain. The disruption of the YLR151c in yeast strain caused ~14-fold increase in the frequency of spontaneous mutation compared to the wild type. Additionally, biochemical analysis indicated that GST-YLR151c fusion protein possessed pyrophosphatase activity for both 7,8-dihydro-8-oxo-2′-deoxyguanosine triphosphate (8-oxo-dGTP) and 1,2-dihydro-2-hydroxy-2′-deoxyadenosine triphosphate (2-OH-dATP). The specific activity of GST-YLR151c for 8-oxo-dGTP was 5.6 × 10⁻³ μM⁻¹ s⁻¹, which was similar to that of RibA, a backup enzyme for MutT in E.coli, but was 150-fold lower than that of hMTH1. From these results, we conclude that YLR151c has an ability to prevent spontaneous mutagenesis via sanitization of oxidized nucleotides, and that it may be the functional homologue of E.coli MutT in S.cerevisiae.     

The structure and luminescence properties of Mn2+/Eu2+/Er3+‐doped MgO‐Ga2O3‐SiO2 glasses and glass‐ceramics

The MgO–Ga₂O₃–SiO₂ glasses and glass‐ceramics samples doped with Eu²⁺/Mn²⁺/Er³⁺ and heated in reductive atmosphere were prepared by the sol–gel method. The structure, morphology and the luminescence properties were studied using X‐ray diffraction, high‐resolution transmission electron microscope, fluorescence spectra, and up‐conversion emission. The luminescence characteristics of doped ions could be influenced by temperature and matrix component. The characteristic emission of Mn²⁺, Eu²⁺ and Er³⁺ were seen and the energy transfer efficiency from Eu²⁺ to Mn²⁺ was enhanced as Mn²⁺ concentration was increased. In addition, the two‐photon process was determined for the Er³⁺‐doped samples.     

Fluorescence detection of carbofuran in aqueous extracts based on dual-emission SiO2@Y2O3:(Eu3+,Tb3+)@MIP core-shell structural nanoparticles

A novel double-windows fluorescence sensor for carbofuran (CF) detection was successfully developed based on rare-earth Eu,Tb-doped Y₂O₃@SiO₂-based molecularly imprinted nanoparticles (MINs) with a multilayer core-shell structure. The recognition process of the MINs for CF was fairly fast and needed only ~8 min to reach a dynamic equilibrium. Interestingly, one fluorescence attenuation window was found with an increase in CF concentration (Q) from 0.1 to 10 μg ml⁻¹ and with a limit of detection (LOD) of 0.04 μg ml⁻¹ at 544 nm belonging to the Tb³⁺ emission, as well as another fluorescence enhanced window within the CF concentration range 10–100 μg ml⁻¹ (LOD = 4 μg ml⁻¹) at 617 nm of Eu³⁺ emission in the dispersed rare-earth-doped MIN colloidal aqueous solution. Luminescence resonance energy transfer from CF to Eu³⁺ and an inner filter effect of CF towards Tb³⁺, as well from the two independent detection windows were clearly observed simultaneously. The competition experiment displayed hardly any marked interference during detection of CF following addition of its analogues (carbaryl, isoprocarb, aldicarb, methomyl, and etofenprox). Moreover, the MINs could also be applied to accurately detect CF in rhubarb and wolfberry samples with recoveries of 85.7–92.2%. This sensing system has high specific recognition and a wide detection range for CF and provides new opportunities for pesticide detection.     

Utilization of fluorescent lanthanide ions for the study of cation binding to extracellular sites in frog skeletal muscle.

The binding of Tb³⁺ ions to frog muscle has been studied, using fluorescence measurements. Kinetic evidence suggests that Tb³⁺ binds to the muscle at multiple sites. Two major classes can be distinguished: One having relatively fast kinetics and saturating within a few minutes; the second type being either very slow (hours) or consisting of an infinite pool. Binding to the first class of sites is readily reversible and is probably extracellular. It is shown that part of the slow type of binding is also reversible, and is also associated with extracellular binding sites.The binding of terbium is antagonized by other metal ions, their order of effectiveness being: La³⁺, Eu³⁺ > Mn²⁺, Hg²⁺ ? Ca²⁺ > Mg²⁺. A comparison with other studies suggests that part of the binding sites are located at the surface membrane of the muscle. Our experiments raise the possibility that part of the slowly exchanged calcium ions may well be of extracellular origin rather than intracellular one. The limitations and advantages of such studies, as a means to probe ion interactions in complex biological systems are discussed.     

Trace amounts of 8-oxo-dGTP in mitochondrial dNTP pools reduce DNA polymerase gamma replication fidelity

Replication of the mitochondrial genome by DNA polymerase γ requires dNTP precursors that are subject to oxidation by reactive oxygen species generated by the mitochondrial respiratory chain. One such oxidation product is 8-oxo-dGTP, which can compete with dTTP for incorporation opposite template adenine to yield A-T to C-G transversions. Recent reports indicate that the ratio of undamaged dGTP to dTTP in mitochondrial dNTP pools from rodent tissues varies from ~1:1 to >100:1. Within this wide range, we report here the proportion of 8-oxo-dGTP in the dNTP pool that would be needed to reduce the replication fidelity of human DNA polymerase γ. When various in vivo mitochondrial dNTP pools reported previously were used here in reactions performed in vitro, 8-oxo-dGTP was readily incorporated opposite template A and the resulting 8-oxo-G-A mismatch was not proofread efficiently by the intrinsic 3′ exonuclease activity of pol γ. At the dNTP ratios reported in rodent tissues, whether highly imbalanced or relatively balanced, the amount of 8-oxo-dGTP needed to reduce fidelity was <1% of dGTP. Moreover, direct measurements reveal that 8-oxo-dGTP is present at such concentrations in the mitochondrial dNTP pools of several rat tissues. The results suggest that oxidized dNTP precursors may contribute to mitochondrial mutagenesis in vivo, which could contribute to mitochondrial dysfunction and disease.     

Fe(III)–resorcylate as a spectrophotometric probe for phospholipid–cation interactions

A simple spectrophotometric microplate assay that allows quantification of the interaction between phospholipids and metal ions or other small cationic compounds has been developed. The assay is based on the competition of the phospholipids for the Fe³⁺ ion in the purple-colored Fe(III)–γ-resorcylate complex and for other cations. To compare the binding affinities of several cation–phospholipid interactions, K_0.5 values were derived from binding curves constructed by determination of the absorbance of the Fe(III)–γ-resorcylate at 490 nm as a function of the cation concentration. The assay was used to analyze the binding of lanthanide ions, calcium ions, and amines (hydrochlorides of ethanolamine, spermidine, and hexyltrimethylammonium chloride) to small unilamellar vesicles (SUVs) and mixed micelles containing anionic lipids such as phosphatidic acid and phosphatidyl-p-nitrophenol. The method was evaluated by fluorescence measurements with Eu³⁺ ions as tracer. Lanthanide ions such as La³⁺ and Ce³⁺ ions showed K_0.5 values smaller by one to two orders of magnitude compared with Ca²⁺ ions. In the presence of increasing amounts of detergents such as Triton X-100, the method also reflected transitions from SUVs to micelles. The binding capacity for metal ions was higher for phospholipid-containing micelles than for the corresponding SUVs.     

A homogeneous resonance energy transfer-based assay to monitor MutS/DNA interactions

Probing the interactions of the DNA mismatch repair protein MutS with altered and damaged DNA is of great interest both for the understanding of the mismatch repair system function and for the development of tools to detect mutations. Here we describe a homogeneous time-resolved fluorescence (HTRF) assay to study the interactions of Escherichia coli MutS protein with various DNA substrates. First, we designed an indirect HTRF assay on a microtiter plate format and demonstrated its general applicability through the analysis of the interactions between MutS and mismatched DNA or DNA containing the most common lesion of the anticancer drug cisplatin. Then we directly labeled MutS with the long-lived fluorescent donor molecule europium tris-bipyridine cryptate ([TBP(Eu³⁺)]) and demonstrated by electrophoretic mobility shift assay that this chemically labeled protein retained DNA mismatch binding property. Consequently, we used [TBP(Eu³⁺)]-MutS to develop a faster and simpler semidirect HTRF assay.