The study of terbium generated bacterirhodopsin

The localization of Terbium (Tb³⁺) cations binding to deionized bacteriorhodopsin (bR) has been studied by using spectroscopic methods. It was found that adding Tb³⁺ cations to deionized bR affects the fluorescence lifetimes of tryptophan (Trp) in bR, the wavelength of fluorescence peak shifts “blue” and the peak value of fluorescence decreases. It was also found that adding one Tb³⁺ cation to deionized bR can restore the purple state from its blue state obviously. The measurements of absorbance, fluorescence and lifetime of fluorescence also show that when more than three Tb³⁺ cations are added, no further changes can be found. It is suggested that one Tb³⁺ specific binding site for the color-controlling is located on the exterior of the bR trimer structure to negatively charged lipids near Trp-10 and Trp-12. Three Tb³⁺ cations binding per bR is needed for the regenerated bR.     

The effect of metal cation binding on the protein, lipid and retinal isomeric ratio in regenerated bacteriorhodopsin of purple membrane

The effect of metal cation binding on bacteriorhodopsin (bR) in purple membrane has been examined using in situ attenuated total reflection-Fourier transform infrared difference spectroscopy in aqueous media. It is known that adding metal cations to deionized bR regenerates the purple state from its blue state and recovers the proton pump function. During this process, infrared spectral changes in the frequency region of 1800-1000 cm-1 are monitored. The results reveal that metal cation binding affects the protein conformation, the retinal isomeric composition as well as lipid head groups. It is also observed that metal cation binding induces conformational changes in the alpha 1-helix region of bR, converting the portion of its alpha 1-helical domain into beta-turn or disordered coil. In addition, the influence of Ho3+ binding on the protein and lipid is observed to be larger than that of Ca2+. These results suggest that some of the metal cation binding sites are on the membrane lipid domain, while others could be on the intrahelical domain or interhelical loops where the Asp and Glu are located (binding with their COO- groups). Our results also suggest that the removal of the C-terminal of bR increase the accessibility of the binding site of metal cations, which affects protein conformational structure. All these observations are discussed in terms of the two proposals given in the literature regarding the metal cation binding sites.     

TIME-RESOLVED RESONANCE RAMAN SPECTRA OF THE PHOTOCYCLE INTERMEDIATES OF ACID AND DEIONIZED BACTERIORHODOPSIN

Abstract— The photocycle of bacteriorhodopsin (bR) and its perturbed forms are investigated by a time-resolved resonance Raman study. These experiments were performed in the C=C stretching and in the fingerprint spectral regions for the acid blue, acid purple and deionized forms of bR.
The main observations are as follows: (1) isomerization of the retinal, from all- trans to 13- cis , occurs in native bR and in all of the acid and deionized perturbed bR species; (2) formation of the early intermediates (the K₆₁₀ and L₅₅₀ analogues) also occur in native bR and in all of the perturbed species; and (3) deprotonation of the protonated Schiff base (PSB), to give the M₄₁₂ type intermediate, occurs in native bR, but is inhibited in all of the perturbed bR species on the time-scale of the native bR photocycle.
The results show that isomerization alone is not a prerequisite for the PSB deprotonation process. The observed photocycle, initiated with retinal isomerization, is found to occur from all- trans to 13- cis in all of the perturbed forms of bR. In addition, the results imply that removal of the cations, of an increase in the hydrogen ion concentration, prevent only the PSB deprotonation process and not the formation of earlier cycle intermediates. Some attention is focused on the two blue forms of bR (acid and deionized) due to the fact that their ground-state absorption maximum, unphotolyzed Raman spectra, and Raman spectra changes during the photocycle are all very similar. The similarities between the acid blue and deionized blue forms in the fingerprint region support previous suggestions that both blue species have nearly the same retinal active site.     

Li2SrSiO4:Ce3+,Tb3+荧光粉的发光特性及Ce3+→Tb3+的能量传递

用固相反应法合成了具有单相的Li2EuSiO4结构的Li2Sr1-x-ySiO4:xCe3+,yTb3+系列样品。荧光光谱研究表明,Li2SrSiO4:Ce3+发射很强的蓝光,最强的激发峰位于360 nm;而Li2SrSiO4:Tb3+发射很强的绿光,最强的激发激发峰位于243 nm,但在350～410 nm的激发非常微弱。在Ce3+,Tb3+共掺杂的样品Li2Sr0.99-ySiO4:0.01Ce3+,yTb3+中,观察到Ce3+对Tb3+的共振能量传递。由于Ce3+对Tb3+能量传递,Tb3+的激发光谱中出现360 nm附近的宽激发峰。控制Tb3+/Ce3+掺杂浓度比可以实现绿蓝双基色的调制。这种双基色的荧光粉有望在紫外激发的白光LED中获得应用。     

Characterization of subdomain IIA binding site of human serum albumin in its native, unfolded, and refolded states using small molecular probes

Subdomain IIA binding site of human serum albumin (HSA) was characterized by examining the change in HSA fluorescence in the native, unfolded, and refolded states. The study was carried out in the absence and presence of small molecular probes using steady-state and time-resolved fluorescence measurements. 2-Pyridone, 3-pyridone, and 4-pyridone bear similar molecular structures to those found in many drugs and are used here as probes. They are found to specifically bind in subdomain IIA and cause a reduction in the fluorescence intensity and lifetime of the Trp-214 residue in native HSA which is located in the same subdomain. The efficiency of energy transfer from Trp-214 fluorescence to the probes was found to depend on the degree of the spectral overlap between the donor's fluorescence and the acceptor's absorption. After probe binding in subdomain IIA, the distance between the donor and acceptor was calculated using Forster theory. The calculated quenching rate constants and binding constants were also shown to depend on the degree of spectral overlap. The results point to a static quenching mechanism operating in the complexes. Denaturation of HSA in the presence of guanidine hydrochloride (GdnHCl) starts at [GdnHCl] > 1.0 M and is complete at [GdnHCl] > or = 6.0 M. Upon unfolding, two fluorescence peaks were observed. One peak was assigned to the fluorescence of Trp-214 in a polar environment, and the other peak was assigned to tyrosine fluorescence. A reduction of the fluorescence intensity of the two peaks upon binding of the probes to the denatured HSA indicates that Tyr-263 in subdomain IIA is one of the tyrosine residues responsible for the second fluorescence peak. The results were confirmed by measuring the fluorescence spectra and lifetimes of denatured HSA at different excitation wavelengths, and of L-tryptophan and L-tyrosine free in buffer. The measured lifetimes of denatured HSA are typical of tryptophan in a polar environment and are slightly reduced upon probe binding. Dilution of the denatured HSA by buffer results in a partial refolding of subdomain IIA. This partial refolding is attributed to some swelling of the binding site caused by water. The swelling prevents a full recovery from the denatured state.     

Laser spectroscopic studies of Tb3+-doped oxyfluoroborate glass

Tb3+-doped oxyfluoroborate glasses have been prepared for different concentrations of Tb. The absorption, fluorescence and photoacoustic spectra of these have been recorded and studied. It is marked that the fluorescence intensity of different fluorescence transitions decreases with the increase of Tb ion concentration in the glass. This quenching at higher concentration is due to the energy transfer among the excited and nearest neighbor unexcited Tb ions in the glass. The lifetime measurement confirms it, as the lifetime of a particular state was found to decrease with the increase of Tb ion concentration in the glass. The mechanism of the energy transfer process was determined to involve quadrupole quadrupole interaction. We have also studied the energy transfer from Tb3+-->Pr3+ when both the rare earths are doped together in the glass. A decrease in the lifetime of the 5D4 level of Tb3+ with the increase of Pr3+ concentration confirms this.     

稀土离子与乳铁蛋白结合的光谱研究

用紫外示差光谱、荧光光谱及圆二色谱等方法研究了Tb³⁺和Eu³⁺在pH7.4的条件下与乳铁蛋白及脱铁乳铁蛋白的结合作用.结果表明,Tb³⁺及Eu³⁺可特异性地结合在脱铁乳铁蛋白的两个Fe³⁺结合部位,但不能从已经结合铁的乳铁蛋白中把铁置换出来.测得Tb³⁺ 与这两个部位结合的条件平衡常数为lgK₁=8.48±0.24和lgK₂=6.72±0.18(25℃,0.10mol/L NaCl, 0.10mol/LHepes,pH=7.4). Tb³⁺在这两个位点结合时,蛋白质的构象发生变化.在 Tb³⁺ 与蛋白质的浓度比低时,构象趋于紧缩,色氨酸残基进入疏水的环境;当Tb³⁺结合得较多时,构象转而开放,色氨酸残基转向亲水性环境.但无论哪种情况,Tb³⁺与脱铁乳铁蛋白的结合都不影响蛋白的二级结构.     

Photocurrent of purple membrane adsorbed onto a thin polymer film: action characteristics of the local anesthetics

The proton pumping activity of bacteriorhodopsin (bR) in the purple membrane adsorbed onto a thin polymer film as a solid support for electrical measurements has been examined in the presence of local anesthetics and 1-alcohols as an anesthetic model. This membrane adsorbed system provided high reproducibility of the photocurrents in bR due to the mechanical and the chemical stability and the electric properties of the thin polymer film. As the concentrations of the local anesthetics increased, the photocurrents generated by the proton pump of bR were cooperatively suppressed and the changes in the photocurrents were reversible. From the dose–response curves for the anesthetics, the concentration (EC₅₀) required for a 50% suppression showed a marked specificity in the order of lidocaine>bupivacaine>tetracaine>dibucaine. The suppression of the photocurrent in bR was more effective for the uncharged form of the local anesthetics than for the charged one. The absorption and fluorescence spectra suggested that the charged form of the anesthetics was bound to the purple membrane surface, while their uncharged form interacted with the hydrophobic portions of the purple membrane interior rather than with the membrane surface. From the dose–response curves for the 1-alcohols, an increase in hydrophobicity in their molecules effectively suppressed the photocurrent of bR. We found that the binding of hydrophobic organic cations such as tetracaine hydrochloride and bupivacaine hydrochloride to the blue membrane with loss of the proton pump, which was prepared by removal of the cations from the purple membrane, could regenerate the proton pumping activity. The photocurrent in bR in the purple membrane adsorbed onto a thin solid film sensitively responded to different local anesthetics.     

Sr2MgSi2O7∶Tb3+，Li+荧光粉的合成和发光机理

采用高温固相法合成Sr2-mMg1-nSi2O7∶mTb3+,nLi+(m=0.03~0.50,n=m)系列荧光粉。使用X射线衍射仪和荧光光谱仪对样品的物相和发光性质进行了表征。在377 nm紫外光激发下,荧光粉的发射光谱呈多谱带发射,主峰位于490 nm,542 nm,590 nm和613 nm处,分别对应于Tb3+的5D4→7FJ(J=6,5,4,3)跃迁发射。调节Tb3+离子掺杂浓度,可实现荧光粉的发光颜色从蓝到白、黄、绿的可调发射;名义组成为Sr1.95Mg0.95Si2O7∶0.05Tb3+,0.05Li+的荧光粉在紫外光(377 nm)激发下发白光,其色坐标(0.322,0.317)接近纯白光(0.33,0.33),是一种潜在的LED用单基质白光荧光粉。     

Binding Constants for Terbium(Ⅲ) with Chicken Apoovotransferrin

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Factors governing the protonation state of cysteines in proteins: an Ab initio/CDM study

The detailed mechanism of metal-cysteine binding is still poorly understood. It is not clear if every metal cation can induce cysteine deprotonation, how the dielectric medium affects this process, and the extent to which other ligands from the metal's first and second coordination shell influence cysteine ionization. It is also not clear if the zinc cation, with its positive charge reduced by charge transfer from the first two bound cysteinates, could still assist deprotonation of the next one or two cysteines in Cys3His and Cys4 zinc-finger cores. Here, we elucidate the factors governing the cysteine protonation state in metal-binding sites, in particular in Zn.Cys4 complexes, using a combined ab initio and continuum dielectric approach. Transition metal dications such as Zn2+ and Cu2+ and trivalent cations such as Al3+ with pronounced ability to accept charge from negatively charged Cys- are predicted to induce cysteine deprotonation, but not "hard" divalent cations such as Mg2+. A high dielectric medium was found to favor cysteine deprotonation, while a low one favored the protonated state. Polarizable ligands in the metal's first shell that can competitively donate charge to the metal cation were found to lower the efficiency of the metal-assisted cysteine deprotonation. The calculations predict that the zinc cation could assist deprotonation of all the cysteines during the folding of Cys4 zinc-finger cores and the [Zn.(Cys-)4]2- state is likely to be preserved in the final folded conformation of the protein provided the binding site is tightly encapsulated by backbone peptide groups or lysine/arginine side chains, which stabilize the ionized cysteine core.     

尖吻蝮蛇毒抗凝血因子Ⅱ的稀土离子荧光探针研究

尖吻蝮蛇毒抗凝血因子(ACF)分子中有两个钙离子结合位点,钙离子对ACF的内源荧光有增强作用,稀土离子(Nd³⁺,Sm³⁺,Eu³⁺,Gd³⁺和Tb³⁺)能取代ACF分子中的钙离子,并对ACF的内源荧光有不同程度的猝灭作用,其中Tb³⁺接受ACF分子中Trp残基传递的能量后,特征荧光增强.稀土离子与ACF荧光滴定表明,ACF分子中有两个稀土离子结合位点,稀土离子和钙离子在ACF分子中两个结合部位是共同的竞争结合部位.ACF与不同稀土离子之间有相近的表观结合常数K₁或K₂.Tb³⁺与RE³⁺(RE=Nd,Sm,Eu或Gd)间线性自由能关系表明,稀土离子与ACF结合时,没有明显的空间效应.ACF分子中的两个结合位点在结构上都有较大的柔性,这种结构柔性为钙离子在ACF与活化凝血因子X的结合反应中起到的促进作用提供了结构基础.     

Determination of trace terbium(III) with N, N', N"-tri(3-indolemethanal)triaminotriethylamine based on a new fluorescence enhancement system.

A new Schiff-base ligand with a tripodal structure, N,N',N"-tri(3-indolemethanal)triaminotriethylamine (TTAIM), was synthesized. Its fluorescence intensity with terbium(III) was increased by about two orders of magnitude in the present of sodium acetate (NaAc). After the adding of the organic solvent dimethyl sulfoxide (DMSO) to the above system, leading to Tb3+ the fluorescence was further enhanced by about 16 fold. The spectrofluorimetric determination of a trace amount of Tb3+ based on this phenomenon was carried out. The excitation and emission wavelengths were 330 nm and 545 nm, respectively. Under the optimal conditions, the fluorescence intensities varied linearly with the concentration of Tb3+ in the range of 5.7 x 10(-11) - 6.3 x 10(-6) mol L(-1) with a detection limit of 5.0 x 10(-11) mol L(-1). The interferences of some rare earth metals and other inorganic ions were described. The method is a selective, sensitive, rapid and simple analytical procedure for the determination of terbium(III) in a high-purity yttrium oxide and synthetic sample. The mechanism for the fluorescence enhancement was also studied.     

The spectral studies on the effect of Glu 101 to the metal binding characteristic of Euplotes octocarinatus centrin

Glu is highly conserved as the first amino acid of E-helix of the EF-hand protein. In this paper, Glu 101, the first amino acid of E-helix of the third EF-hand motif in Euplotes octocarinatus centrin (EoCen) was mutated to be Lys by the method of site direct mutation. Tb3+ and TNS were used as fluorescence probes in the study of the effect of this mutation to the metal binding characteristic of EoCen by fluorescence spectra. Results indicate that compared with EoCen, the mutation protein (E101K) displays a different Tb3+ binding characteristic and an increased hydrophobic exposure surface. Polyacrylamide gels electrophoresis indicated that the electrophoretic mobilities of EoCen and E101K are distinctly different. It can be deduced that the conformation of EoCen has been altered by this mutation. The general conditional binding constant of Tb3+ to the three loops of EF-hand sites I-III in E101K was calculated to be (5.64+/-0.57)x10(5)M(-1) according to the modified equation of the single binding process.     

Effect of metal ion exchange on the photocurrent response from bacteriorhodopsin on tin oxide electrodes

The transient photocurrent response from bacteriorhodopsin (bR) on tin oxide electrodes was strongly influenced by metal ions bound to bR molecules. The photocurrent polarity reversal pH, which corresponded to the pH value for the reversal of the proton release/uptake sequence in the bR photocycle, of cation-substituted purple membrane (PM) was shifted to lower pH with the increase in the cation affinities to carboxyl groups and a close correlation was noted between the two values. This suggests that the metal ion present in the extracellular region of a bR molecule modulates the pK(a) of proton release groups of bR by stabilizing the ionized state of the proton-releasing glutamic acids. The behavior of photocurrents at light-off in alkaline media, reflecting the proton uptake by bR, was unchanged by binding monovalent (Na(+) and K(+)) or divalent cations (Mg(2+) and Ca(2+)), but was drastically changed by binding La(3+) ions. This can be explained by invoking a substantial slowing of the proton uptake process in the presence of La(3+).     

顺二氨二水合铂(Ⅱ)与膜磷脂及膜蛋白相互作用的荧光光谱研究

用Tb~(3+)分别对用和未用顺二氨二水合铂(Ⅱ)(AAP)处理的红细胞膜和磷酰胆碱(PC)脂质体进行荧光滴定,测Tb~(3+)荧光。用AAP滴定红细胞膜,测蛋白质自身荧光变化。用间接Scatchard法求结合参数。表明蛋白质及磷脂均与AAP结合,蛋白质结合能力较强。     

Lanthanide luminescent switches: modulation of the luminescence of bis-macrocyclic based Tb(III) conjugates in water by H+, Na+ and K+

The synthesis of four bis-macrocyclic conjugates made from the coupling of either diaza-15-crown-5 ethers (1 and 3) and diaza-18-crown-6 ethers (2 and 4) to either amide or carboxylate functionalized cyclen (1,4,7,10-tetraazacyclododecane), and their corresponding cationic Tb(III) complexes, Tb-1, Tb-2, and neutral complexes Tb-3 and Tb-4 are described. The effect on the ground, singlet excited states and the Tb(III) emission, was investigated either as a function of pH or the concentration of several Group I and II cations, upon excitation at 300 nm. The ground state and singlet excited states of the Tb(III) complexes were found to be modulated by ions such as H+, Na+ or K+, signifying the recognition of these ions by the crown ether receptors. In acidic media, below pH 4, the Tb(III) emission was highly pH sensitive, gradually increasing with large orders of magnitude of luminescence enhancements. For Tb-1 and Tb-2 complexes, the Tb(III) emission was also "switched on" in alkaline media above pH 8. At pH 7.4, the recognition of Na+ or K+ also gave rise to a significant change in the Tb(III) emission due to the modulation of the antenna-receptor moieties by these ions. For Tb-1 and Tb-3 the largest changes were seen for Na+, whereas for Tb-2 and Tb-4 the largest changes were seen for K+.     

The Special Case of the Spectral Emission of a Tb3+ Mono Metal Complex

The fine structure in the spectral lines of the visible fluorescence of Tb³⁺ complexes are replaced by a single peak in the case of a singular molecular complex Tb(H₃PTC)₃, where H₄PTC represents perylene-3,4,9,10-tetracarboxylic acid, and its emission wavelength depends on the film thickness. This single peak challenges the old creed that the f-orbital electrons of Tb³⁺ are always protected from the influence of the surrounding atoms. We perform density functional theory calculations to show that the wavefunction of the ground state is localized and in addition, spin-polarized, and this facilitates fluorescent transitions under UV to the first excited state instead of the fundamental state. We discuss the possibility of making a spintronic device with the molecule, Tb(H₃PTC)₃.     

氧氟沙星与脲诱导牛血清白蛋白结合的机制研究

摘要 利用荧光光谱和紫外光谱研究了脲（Urea）对牛血清白蛋白（BSA）结构的影响以及氧氟沙星（Oflxacin）与脲诱导的BSA结合的情况。结果显示：Urea诱导BSA变性历经两步、三态过程,且伴随中间态的形成。随着Urea浓度的增大,BSA荧光强度降低并先蓝移（344 nm～336 nm）,后又红移至350 nm。Urea浓度在4.6～5.2 mol/L范围时,Oflx对BSA中间态有强的猝灭作用（KQ=10.46×104 L/mol, Urea 4.8 mol/L）和较大的结合常数（KA=3.8807×105 L/mol, Urea 4.8 mol/L）,但是结合位点数小（n=0.76, Urea 5.0 mol/L）,能量传递效率低（E=0.3002, Urea 4.8 mol/L）。同步荧光光谱显示：Urea诱导BSA去折叠时,Trp-212残基微环境并未发生改变,而Tyr的最大荧光发射峰蓝移,Oflx的加入诱导Trp-212的微环境更具疏水性。Oflx加速了Urea对BSA的失活作用。