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.     

Metal ions-induced conformational change of P23 by using TNS as fluorescence probe

In 10 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (Hepes), pH 7.4, 25 degrees C, the conformational change of the truncated form of ciliate Euplotes Octocarinatus centrin (P23) induced by metal ions were investigated using 2-p-toluidinylnaphthalene-6-sulfonate (TNS) as a probe. The results show that upon metal ions binding, P23 undergo a conformational change and the contributions to the conformational change from the two EF-hands are different, and Tb3+ has more larger influence than Ca2+ with the same concentration metal ions, which provide possible the evidence that the different EF-hands play distinct biological functions. Meanwhile, the conditional binding constants of TNS and Ca2-loaded or Tb2-loaded P23 were obtained, K (Ca2-P23+TNS)=(7.49+/-0.88)x10(5) mol-1 L, K (Tb2-P23+TNS)=(8.24+/-0.49)x10(5) mol-1 L.     

ESR and optical study of Cu2+ doped calcium malonate dihydrate

The ESR study of Cu(2+) doped calcium malonate dihydrate has been done at room temperature. Four magnetically in-equivalent sites for Cu(2+) have been observed. The spin-Hamiltonian parameters evaluated with the fitting of spectra to rhombic symmetry crystalline field are for Cu(2+) site (I): g(x)=2.0963+/-0.0002, g(y)=2.1316+/-0.0002, g(z)=2.4137+/-0.0002, A(x)=(32+/-2)x10(-4)cm(-1), A(y)=(34+/-2)x10(-4)cm(-1), A(z)=(49+/-2)x10(-4)cm(-1), for site (II): g(x)=2.0668+/-0.0002, g(y)=2.0800+/-0.0002, g(z)=2.3561+/-0.0002, A(x)=(34+/-2)x10(-4)cm(-1), A(y)=(36+/-2)x10(-4)cm(-1), A(z)=(51+/-2)x10(-4)cm(-1), for site (III): g(x)=2.0438+/-0.0002, g(y)=2.0623+/-0.0002, g(z)=2.2821+/-0.0002, A(x)=(34+/-2)x10(-4)cm(-1), A(y)=(36+/-2)x10(-4)cm(-1), A(z)=(53+/-2)x10(-4)cm(-1), and for site (IV): g(x)=2.0063+/-0.0002, g(y)=2.0241+/-0.0002, g(z)=2.2357+/-0.0002, A(x)=(35+/-2)x10(-4)cm(-1), A(y)=(37+/-2)x10(-4)cm(-1), A(z)=(54+/-2)x10(-4)cm(-1). The ground state wave function of Cu(2+) has also been determined. The g-anisotropy has been estimated and compared with the experimental value. Further with the help of optical study the nature of bonding of metal ion with different ligands in the complex has been discussed.     

The quantum cutting of Tb(3+) in Ca(6)Ln(2)Na(2)(PO(4))(6)F(2) (Ln = Gd, La) under VUV-UV excitation: with and without Gd(3+)

The VUV-vis spectroscopic properties of Tb(3+) activated fluoro-apatite phosphors Ca(6)Ln(2-x)Tb(x)Na(2)(PO(4))(6)F(2) (Ln = Gd, La) were studied. The results show that phosphors Ca(6)Gd(2-x)Tb(x)Na(2)(PO(4))(6)F(2) with Gd(3+) ions as sensitizers have intense absorption in the VUV range. The emission color of both phosphors can be tuned from blue to green by changing the doping concentration of Tb(3+) under 172 nm excitation. The visible quantum cutting (QC) via cross relaxation between Tb(3+) ions was observed in cases with and without Gd(3+). Though QC can be realized in phosphors Ca(6)La(2-x)Tb(x)Na(2)(PO(4))(6)F(2), we found that Gd(3+)-containg phosphors have a higher QC efficiency, confirming that the Gd(3+) ion indeed plays an important role during the quantum cutting process. In addition, the energy transfer process from Gd(3+) to Tb(3+) as well as (5)D(3)-(5)D(4) cross relaxation was investigated and discussed in terms of luminescence spectra and decay curves.     

Characterization of melittin binding to Euplotes octocarinatus centrin

In the presence of 1.0mM Ca(2+), the interaction between Euplotes octocarinatus centrin (EoCen) and melittin (ME) was studied by means of fluorescence spectra. In 0.1M N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (Hepes) and 150mM NaCl at pH 7.4, fluorescence peak of ME was observed at about 353nm indicating that micro-environment of Tryptophan (Trp) residue in ME was hydrophilic. With the addition of 3.2x10(-4)M calcium saturated EoCen (holoEoCen), the peak of ME was blue-shifted to 339nm, which may be resulted from micro-environmental changes of the peptide. At the same time, fluorescence emission of ME was increased significantly suggesting that new complex of ME-holoEoCen was formed under the experimental conditions. Based on the fluorescence titration curves, the 1:1 stoichiometric ratio of holoEoCen to ME was confirmed. In addition, the conditional binding constant of holoEoCen with ME was calculated to be logK(ME-holoEoCen)=6.59+/-0.14.     

Analysis of the role of Mg²⁺ on conformational change and target recognition by ciliate Euplotes octocarinatus centrin

The binding of Mg(2+) with the Euplotes octocarinatus centrin (EoCen) and the effect of Mg(2+) on the binding of EoCen with the peptide melittin were examined by spectroscopic methods. In this study, it was found that Mg(2+) may bind with Ca(2+)-binding sites, at least partly, on EoCen, which displays ～10-fold weaker affinity than Ca(2+). In the presence of Mg(2+), Ca(2+)-saturated EoCen undergoes significant conformational changes resulting in decreased exposure of hydrophobic surfaces on the protein. Additionally, excess Mg(2+) did not change the stoichiometry, but rather reduced the affinity of EoCen to melittin. The Mg(2+)-dependent decrease in the affinities of EoCen to melittin is an intrinsic property of Mg(2+), rather than a nonspecific ionic effect. The inhibitory effect of Mg(2+) on the formation of complexes between EoCen and melittin may contribute to the specificity of EoCen in target activation in response to cellular Ca(2+) concentration fluctuations.     

Spectral study on the binding of gadolinium ions with apoovotransferrin

The binding of Gd3+ ion to apoovotransferrin (apoOTf) was monitored by means of UV difference spectra in 0.01M Hepes, pH 7.4 at 25 degrees C. Used 2-p-toluidinylnaphthalene-6-sulfonate (TNS) as fluorescence probe the conformational changes of protein were studied while gadolinium ions bound to apoOTf. The results show that Gd3+ binding produces peaks at 244 and 294 nm that is the characteristic of binding at the apoOTf specific metal-binding sites. At 244 nm the molar absorptivity of Gd-apoOTf complex is (1.99+/-0.17)x10(4)cm(-1)M(-1). The apparent binding constants for the complexes of Gd3+ with apoovotransferrin are logK(1)=7.61+/-0.14 and logK(2)=4.96+/-0.26. A very large conformational change of apoovotransferrin appears when Gd3+ is bound to the N-terminal binding site. When Gd3+ is bound to C-terminal binding site there is less conformational change.     

UV and fluorescence spectral changes induced by neodymium binding of N,N'-ethylenebis[2-(o-hydroxyphenolic)glycine] and N,N'-di(2-hydroxybenzyl)ethylenediamine-N,N' diacetic acid

In 0.01 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes), pH 7.4 and room temperature, the binding of neodymium to N,N'-ethylenebis[2-(o-hydroxyphenolic)glycine] (EHPG), or N,N'-di(2-hydroxybenzyl)ethylenediamine-N,N' diacetic acid (HBED) had been studied from 210 to 330 nm by means of difference UV spectra. Two peaks at 240 and 292 nm appear in difference UV spectra after neodymium binding to EHPG or HBED. The 1:1 stable complex can be confirmed from spectral titration curves. The molar extinction coefficient of Nd-EHPG and Nd-HBED complexes are Deltaepsilon(Nd-EHPG)=(12.93+/-0.21) x 10(3)cm(-1)M(-1), Deltaepsilon(Nd-HBED)=(14.45+/-0.51) x 10(5)cm(-1)M(-1) at 240 nm, respectively. Using EDTA as a competitor, the conditional equilibrium constants of the complexes are logK(Nd-EHPG)=11.89+/-0.09 and logK(Nd-HBED)=12.19+/-0.15, respectively. At the same conditions, fluorescence measurements show that neodymium binding to EHPG leads to a quenching of the fluorescence of EHPG at near 310 nm. However, there is no obvious fluorescence change of HBED at 318 nm with the binding of neodymium to HBED.     

Spectroscopic properties and thermal stability of Er3+ -doped TeO2-B2O3-Nb2O5-ZnO glass for potential WDM amplifier

A series of novel 70TeO2-(15-x)B2O3-xNb2O5-15ZnO-1wt.% Er2O3 (TBN x=0, 3, 6, 9, 12 and 15 mol%) tellurite glasses were prepared. The thermal stability, absorption spectra, emission spectra, and the lifetime of the (4)I(13/2) level of Er(3+) ions were measured and investigated. Three Judd-Ofelt intensity parameters Omega(t) (t=2, 4 and 6) (Omega(2)=(5.42-6.76)x10(-20)cm(2); Omega(4)=(1.37-1.73)x10(-20)cm(2); Omega(6)=(0.70-0.94)x10(-20)cm(2)) of Er(3+) ions were calculated by Judd-Ofelt theory. It is found that the Omega(6) first increases with the increase of Nb2O5 content from 0 to 6 mol% and then decreases, which is mainly affected by the number of non-bridging oxygen ions of the glass network. The high peak of stimulated emission cross-section (sigma(e)(peak)=(0.77-0.91)x10(-20)cm(2)) of Er(3+): (4)I(13/2)-->(4)I(15/2) transition were obtained according to McCumber theory and broad full width at half maximum (FWHM=65-73 nm) of the (4)I(13/2)-->(4)I(15/2) transition of Er(3+) ions were measured. The results indicate that these new TBN glasses can be used as a candidate host material for potential broadband optical amplifiers.     

Rate coefficients for the reaction of OH with (E)-2-pentenal, (E)-2-hexenal, and (E)-2-heptenal

Rate coefficients for the gas-phase reaction of the OH radical with (E)-2-pentenal (CH(3)CH(2)CH[double bond]CHCHO), (E)-2-hexenal (CH(3)(CH(2))(2)CH[double bond]CHCHO), and (E)-2-heptenal (CH(3)(CH(2))(3)CH[double bond]CHCHO), a series of unsaturated aldehydes, over the temperature range 244-374 K at pressures between 23 and 150 Torr (He, N(2)) are reported. Rate coefficients were measured under pseudo-first-order conditions in OH with OH radicals produced via pulsed laser photolysis of HNO(3) or H(2)O(2) at 248 nm and detected by pulsed laser-induced fluorescence. The rate coefficients were independent of pressure and the room temperature rate coefficients and Arrhenius expressions obtained are (cm(3) molecule(-1) s(-1) units): k(1)(297 K)=(4.3 +/- 0.6)x 10(-11), k(1)(T)=(7.9 +/- 1.2)x 10(-12) exp[(510 +/- 20)/T]; k(2)(297 K)=(4.4 +/- 0.5)x 10(-11), k(2)(T)=(7.5 +/- 1.1)x 10(-12) exp[(520 +/- 30)/T]; and k(3)(297 K)=(4.4 +/- 0.7)x 10(-11), k(3)(T)=(9.7 +/- 1.5)x 10(-12) exp[(450 +/- 20)/T] for (E)-2-pentenal, (E)-2-hexenal and (E)-2-heptenal, respectively. The quoted uncertainties are 2sigma(95% confidence level) and include estimated systematic errors. Rate coefficients are compared with previously published room temperature values and the discrepancies are discussed. The atmospheric degradation of unsaturated aldehydes is also discussed.     

Stabilization of calcium- and terbium-carboxylate bonds by NH...O hydrogen bonds in a mononuclear complex: a functional model of the active site of calcium-binding proteins

Novel benzoic acid ligands with bulky amide groups at the ortho position, 2,6-(MeCONH)(2)C(6)H(3)CO(2)H (1) and 2,6-(t-BuCONH)(2)C(6)H(3)CO(2)H (2), and their tris- and tetrakis(carboxylate) complexes with Ca(II) and Tb(III) ions, (NEt(4))(2)[Ca(II)[O(2)C-2,6-(t-BuCONH)(2)C(6)H(3)](4)] (4), [Tb[O(2)C-2,6-(t-BuNHCO)(2)C(6)H(3)](3)(H(2)O)(3)]] (5), and (NMe)(4)[Tb[O(2)C-2,6-(t-BuNHCO)(2)C(6)H(3)](4)(thf)] (6), were synthesized. The formation of the NH...O hydrogen bonds between the amide NH and carboxylate for 2, (NEt(4))[2,6-(t-BuCONH)(2)C(6)H(3)CO(2)] (3), and 4 was determined by (1)H NMR spectroscopy in solution and in the solid state (CRAMPS, IR). The ligand exchange reactions were attempted between 4 and a large excess of 2,4,6- Me(3)C(6)H(3)CO(2)H in chloroform-d solution; however, exchange reaction did not take place, indicating that the Ca(II) ions bound strongly to the carboxylate in 4. The Ca(II) ion binding properties with the benzoate derivatives were also examined using Tb(III) ion as a fluorescence probe. These results indicate that the NH...O hydrogen bonding between the amide NH and the oxygen atom of the carboxylate contributes to strong Ca(II) binding and prevents the dissociation of the calcium-carboxylate bond. The X-ray structural analyses of these complexes revealed that the NH.O hydrogen-bonded carboxylate ligands prefer the chelate-type coordination and create a mononuclear [Ca(O(2)CR)(4)](2)(-) or [Tb(O(2)CR)(4)](-) core with anionic charge, which is known only in the active site of calcium-binding proteins.     

Spectroscopic analysis of the interaction between gallium(III) and apoovotransferrin

Ovotransferrin is a main member of transferrin family and has a dual role in both the transport of iron and antibacterial function. Gallium-67 is widely used as an imaging agent for tumors. It has been reported that Ga(3+) can bind to apoovotransferrin at two sites, one in the N-terminal lobe and another in the C-terminal lobe. However, several details of the interaction between Ga(3+) and apoOTf remain unclear. Here, we report detailed investigations into the interactions of Ga(3+) with apoovotransferrin at the molecular level. First, the characteristics of Ga(3+) binding to apoovotransferrin were analyzed using UV difference spectra. The results show that Ga(3+) prefers to bind to the N-terminal site rather than the C-terminal site under the experimental conditions. Effective stability constants of logK(N)=18.88+/-0.24 and logK(C)=17.65+/-0.12 were determined. Second, conformational changes in apoovotransferrin during Ga(3+) binding were studied using 2-p-toluidinylnaphthalene-6-sulfonate (TNS) as a fluorescence probe. Apoovotransferrin undergoes a large conformational change when Ga(3+) binds to the N-terminal site, and a smaller conformational change when the ion binds to the C-terminal site. UV difference spectra were also used to measure the rate at which EDTA removes Ga(3+) from ovotransferrin carrying one Ga(3+) at the N-terminal site. Ga(3+) removal from the N-terminal binding site follows simple saturation kinetics.     

Luminescence behavior of a water soluble calix[4]arene derivative complex with terbium ion(III) in gelation solution

The complexation luminescence behavior of a water soluble calix[4]arene derivative, 5,11,17,23-tetra-sulfonate-25,26,27,28-tetra-carboxymethoxycalix[4]arene (L) with lanthanoid ion (Tb(3+)) has been investigated in gelation solution at 25 degrees C by using UV-vis and fluorescence spectra. The results obtained indicated that the water soluble calix[4]arene derivative can form an efficient energy transfer complex with terbium ion(III). The fluorescence of L x Tb(3+)complex is partially quenched by gelatin in gelation solution. The quenching intensity is related to the concentration and the hydrolysis degree of gelatin. Absorption and fluorescence spectra analysis show that the -COO(-) groups on gelatin have a definite binding ability to Tb(3+), and then, gelatin could compete binding with calix[4]arene derivative upon complexation with Tb(3+), leading to the relative fluorescence quenching of the formation complex of terbium(III) ion with calix[4]arene derivative.     

An extremely stable host-guest complex that functions as a fluorescence probe for calcium ions

Herein, we report a crown ether based molecular cage that forms extremely stable supramolecular complexes with dimethyldiazapyrenium (DMDAP) ions in CD(3)CN through the collaboration of multiple weak C-HO hydrogen bonds. The very strong binding affinity in this host-guest system allows the molecular cage to bleach the fluorescence signal of DMDAP substantially in equimolar solutions at concentrations as low as 1 x 10(-5) M. Remarkably, a 1x10(-5) M equimolar solution of the molecular cage and DMDAP is highly selective toward Ca(2+) ions-relative to other biologically important Li(+), Na(+), K(+), and Mg(2+) ions-and causes a substantial increase in the fluorescence intensity of the solution. As a result, this molecular cage/DMDAP complex behaves as a supramolecular fluorescence probe for the detection of Ca(2+) ions in solution.     

Spectral characterization of a newly synthesized fluorescent semicarbazone derivative and its usage as a selective fiber optic sensor for copper(II)

In this work photoluminescent properties of highly Cu(2+) selective organic fluoroionophore, semicarbazone derivative; bis(naphtho[2,1-b]furan-2-yl)methanone semicarbazone (BNF) was investigated in different solvents (dichloromethane, tetrahydrofuran, toluene and ethanol) and in polymer matrices of polyvinylchloride (PVC) and ethyl cellulose (EC) by absorption and emission spectrometry. The BNF derivative displayed enhanced fluorescence emission quantum yield, Q(f)=6.1 x 10(-2) and molar extinction coefficient, epsilon=29,000+/-65 cm(-1)M(-1) in immobilized PVC matrix, compared to 2.6 x 10(-3) and 24,573+/-115 in ethanol solution. The offered sensor exhibited remarkable fluorescence intensity quenching upon exposure to Cu(2+) ions at pH 4.0 in the concentration range of 1.0 x 10(-9) to 3.0 x 10(-4)M [Cu(2+)] while the effects of the responding ions (Ca(2+), Hg(+), Pb(2+), Al(3+), Cr(3+), Mn(2+), Mg(2+), Sn(2+), Cd(2+), Co(2+) and Ni(2+)) were less pronounced.     

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.     

Relative and absolute kinetic studies of 2-butanol and related alcohols with tropospheric Cl atoms

A newly constructed chamber/Fourier transform infrared system was used to determine the relative rate coefficient, k(i), for the gas-phase reaction of Cl atoms with 2-butanol (k(1)), 2-methyl-2-butanol (k(2)), 3-methyl-2-butanol (k(3)), 2,3-dimethyl-2-butanol (k(4)) and 2-pentanol (k(5)). Experiments were performed at (298 +/- 2) K, in 740 Torr total pressure of synthetic air, and the measured rate coefficients were, in cm(3) molecule(-1) s(-1) units (+/-2sigma): k(1)=(1.32 +/- 0.14) x 10(-10), k(2)=(7.0 +/- 2.2) x 10(-11), k(3)=(1.17 +/- 0.14) x 10(-10), k(4)=(1.03 +/- 0.17) x 10(-10) and k(5)=(2.18 +/- 0.36) x 10(-10), respectively. Also, all the above rate coefficients (except for 2-pentanol) were investigated as a function of temperature (267-384 K) by pulsed laser photolysis-resonance fluorescence (PLP-RF). The obtained kinetic data were used to derive the Arrhenius expressions: k(1)(T)=(6.16 +/- 0.58) x 10(-11)exp[(174 +/- 58)/T], k(2)(T)=(2.48 +/- 0.17) x 10(-11)exp[(328 +/- 42)/T], k(3)(T)=(6.29 +/- 0.57) x 10(-11)exp[(192 +/- 56)/T], and k(4)(T)=(4.80 +/- 0.43) x 10(-11)exp[(221 +/- 56)/T](in units of cm(3) molecule(-1) s(-1) and +/-sigma). Results and mechanism are discussed and compared with the reported reactivity with OH radicals. Some atmospheric implications derived from this study are also reported.     

1H and (113)Cd NMR Investigations of Cd(2+) and Zn(2+) Binding Sites on Serum Albumin: Competition with Ca(2+), Ni(2+), Cu(2+), and Zn(2+)

1H and (113)Cd NMR studies are used to investigate the Cd(2+) binding sites on serum albumin (67 kDa) in competition with other metal ions. A wide range of mammalian serum albumins possess two similar strong Cd(2+) binding sites (site A 113-124 ppm; site B 24-28 ppm). The two strong sites are shown not to involve the free thiol at Cys34. Ca(2+) influences the binding of Cd(2+) to isolated human albumin, and similar effects due to endogenous Ca(2+) are observed for intact human blood serum. (1)H NMR studies show that the same two His residues of human serum albumin are perturbed by Zn(2+) and Cd(2+) binding alike. Zn(2+) displaces Cd(2+) from site A which leads to Cd(2+) occupation of a third site (C, 45 ppm). The N-terminus of HSA is not the locus of the two strong Cd(2+) binding sites, in contrast to Cu(2+) and Ni(2+). After saturation of the N-terminal binding site, Cu(2+) or Ni(2+) also displaces Cd(2+) from site A to site C. The effect of pH on Cd(2+) binding is described. A common Cd(2+)/Zn(2+) binding site (site A) involving interdomain His residues is discussed.     

Binding Constants for Terbium(Ⅲ) with Chicken Apoovotransferrin

ＩｎｔｒｏｄｕｃｔｉｏｎＴｈｅｔｒａｎｓｆｅｒｒｉｎｓａｒｅａｓｕｐｅｒｆａｍｉｌｙｏｆｍｅｔａｌ ｂｉｎｄｉｎｇｐｒｏｔｅｉｎｓ ,ｗｈｉｃｈａｒｅｓｉｎｇｌｅ ｃｈａｉｎｓ ,80ｋＤａｇｌｙｃｏｐｒｏｔｅｉｎｓ ,ｃｈａｒａｃｔｅｒｉｚｅｄｂｙｔｈｅｉｒａｂｉｌｉｔｙｔｏｂｉｎｄｉｒｏｎｔｉｇｈｔｌｙ(ａｎｅｆｆｅｃｔｉｖｅｂｉｎｄｉｎｇｃｏｎ ｓｔａｎｔｉｓａｂｏｕｔ 1 0 2 0 .7) ,ｂｕｔｒｅｖｅｒｓｉｂｌｙ[1,2 ] .Ｓｅｒｕｍｔｒａｎｓｆｅｒｒｉｎ ,ｆｏｕｎｄｉｎｂｌｏｏｄａｎｄｅｘｔｒａｃｅｌｌｕｌ…     

八肋游仆虫中心蛋白N端半分子Loop区首个氨基酸的作用

本文通过分子生物学方法将八肋游仆虫中心蛋白N端半分子loop区的首个氨基酸,天冬氨酸Asp37和Asp73,分别突变为带相反电荷的赖氨酸。使用铽敏化荧光、TNS疏水探针研究了八肋游仆虫中心蛋白N端半分子loop区的首个氨基酸的作用。结果表明：当中心蛋白loop Ⅰ区37位的天冬氨酸突变为赖氨酸后,loop Ⅰ丧失了金属离子结合能力,进而影响了中心蛋白依赖于金属离子的构象变化;而loop Ⅱ区73位的天冬氨酸突变为赖氨酸后仍保持金属离子结合能力,依赖于金属离子的构象变化减小。中心蛋白发挥大部分生物功能都依赖于金属离子,这就表明loop Ⅰ区37位的天冬氨酸在中心蛋白发挥生物功能时起着重要作用,是不可缺少的。在10 mmol·L^-1 Hepes、pH 7.4、20 mmol·L^-1 KCl条件下,八肋游仆虫中心蛋白N端半分子loop Ⅱ与金属离子Tb³⁺和Ca²⁺的结合常数分别为：K_Ⅱ(Tb³⁺)=(8.31±0.18)×10⁴ L·mol^-1和K_Ⅱ(Ca²⁺)=(0.94±0.12)×10² L·mol^-1,中心蛋白N端半分子的两个金属结合部位结合能力顺序为：Ⅰ>Ⅱ。