气相中碱金属离子与丝氨酸、 亮氨酸和赖氨酸五肽复合物的裂解反应

为了探索金属离子对含有不同侧链的多肽气相解离的影响, 采用质谱法研究了碱金属离子Li⁺, Na⁺, K⁺, Rb⁺和Cs⁺分别与丝氨酸、 亮氨酸和赖氨酸五肽(分别简写为S5, L5和K5)形成的复合物的裂解反应. 质谱定性结果表明, 5种碱金属离子均可以在气相中与丝氨酸、 亮氨酸和赖氨酸五肽形成配合比为1∶1 和2∶1的非共价复合物; 竞争反应结果表明, 随着碱金属离子半径的增加, 它们与3种五肽的结合能力逐渐减弱. 质谱定量结果表明, K⁺与丝氨酸、 亮氨酸和赖氨酸五肽复合物的结合常数分别为8.94×10⁴, 2.83×10⁴和2.50×10³ L/mol, 表明K⁺与五肽复合物的结合强度按照丝氨酸、 亮氨酸和赖氨酸的顺序依次减小. 含不同侧链碱金属离子-五肽复合物的碰撞诱导解离结果表明, 复合物的碎裂主要发生在骨架上, 丝氨酸五肽复合物最易碎裂, 亮氨酸五肽复合物其次, 赖氨酸五肽复合物则较难碎裂, 且3种复合物的侧链断裂情况也呈现明显差异. 此外, 研究了Na⁺与亮氨酸五肽复合物所产生的碎片离子, 分析了不同离子之间的来源关系, 并以Dunbar的复合物理论模型为依据, 推测在碎裂过程中, 碱金属离子可能向五肽的碳端或氮端偏移. 质谱碎片分析结果表明, 在2∶1的非共价复合物中, 第一个碱金属离子与五肽上4个酰胺键的羰基结合, 第二个碱金属离子与五肽的羧基氧原子结合.     

Tb3+ as Fluorescent Probe in the Study of the Interaction of Other Lanthanide Ions with Trichosanthin

The interaction of lanthanide (Ln³⁺)ions with trchosanthin(TCS)was investi-gated using Tb³⁺ as fluorescent probe.The metal-binding sites of the protein were probed by means of adding the Tb³⁺ into the protein solutions.If other Ln³⁺ ions exist in the Tb³⁺ protein complex system, they would compete against Tb³⁺ in the binding sites of the protein, and so the fluorescence intensity of Tb³⁺ decreased,which is called quenching effect.The quencliing effect is related to the ionic radii of the Ln³⁺ ions and energy transfer from Tb³⁺ protein to Ln³⁺ ions.Based on the ex-perimental results,a schematic of the energy transfer from protein-Tb³⁺ complex to protein-Ln³⁺ complex is suggested.     

USE OF THE FLUORESCENT LANTHANIDE Tb3+ AS A PROBE FOR CATION-BINDING SITES ASSOCIATED WITH ISOLATED CHLOROPLAST THYLAKOID MEMBRANES

Abstract— The fluorescent properties of the rare-earth ion, Tb³⁺ have been utilized to probe the nature of cation-binding sites associated with thylakoid membranes. At low concentrations (< 100μ M ), Tb³⁺ was observed to inhibit the increase in chlorophyll a fluorescence normally seen on adding 5 m M MgCl₂ (or 100 m M NaCl) to isolated, broken chloroplasts. We also observed under these conditions, the appearance of a new band around 280 nm in the excitation spectrum of Tb³⁺ ion fluorescence. However, similar changes in Tb³⁺ fluorescence could be observed in the presence of a membrane-free preparation of chloroplast coupling factor protein (CF₁). From this and other results it is concluded that changes in Tb³⁺ fluorescence reflect an association of the ion with CF₁ followed by intermolecular transfer of excitation energy from protein ligands (possibly un-ionized tyrosine residues) to the lanthanide. The interaction of Tb³⁺ with sites which control chlorophyll a fluorescence does not seem to modify Tb³⁺ fluorescence, suggesting that in this case, simple membrane-bound ligands such as carboxyl or phosphate groups are involved.     

4-甲基苯并-15-冠-5对某些镧系放射性核素的萃取

本文研究了4-甲基苯并-15-冠-5三氯甲烷溶液从苦味酸水溶液中对某些镧系放射性核素(¹⁴⁴Ce、¹⁴⁷Nd、^152+154Eu、¹⁷⁰Tm和¹⁶⁹Yb)的萃取。从斜率分析得出,被萃取状态为1:2:3(金属离子/冠醚/苦味酸根)络合物。在25℃,测定了三价镧系元素的苹取常数(K_ex),得到下列次序:Nd³⁺>Eu³⁺>Ce³⁺>Tm³⁺>Yb³⁺。结果表明,Nd³⁺的离子大小(r=0.995)比其它镧系元素可能更接近4-甲基苯并-15-冠-5环孔的大小。 为了将镧系元素与锕系元素作比较,还测定了4-甲基苯并-15-冠-5对Th⁴⁺、UO₂²⁺和Am³⁺的萃取率,所得到的萃取率次序为Th⁴⁺>Am³⁺UO₂²⁺。实验证明,4-甲基苯并-15-冠-5将可能是溶剂萃取法分离铀和钍的有用试剂。 溶剂和添加剂影响萃取实验表明,在有机相添加正丁醇或异戊醇,或者以高极性的溶剂(例如,硝基苯)替代三氯甲烷,都能显著地增加对镧系元素的萃取。     

VISIBLE LIGHT IRRADIATION OF HUMAN AND BOVINE SERUM ALBUMIN-BILIRUBIN COMPLEX*

Abstract— The UV absorption and the fluorescence emission spectra of both bovine (BSA) and human (HSA) serum albumin underwent noticeable changes upon irradiation of their 1:1 complexes with bilirubin; both these phenomena are suggestive of the photosensitized modification of aromatic amino acid residues. Amino acid analysis showed that after relatively short irradiation times of both albumins, only histidyl and tryptophyl residues appeared to be affected to a significant extent. After 60min of irradiation, some decrease in the tyrosine content was also observed, especially for HSA.
Conformational studies, obtained by exposing unirradiated and irradiated BSA and HSA to denaturing agents, showed that the three-dimensional organization of the 15 min irradiated samples was slightly different from that of the native proteins. On the other hand, after 15 min of irradiation, the association constant of the bilirubin-albumin complexes decreased from 2.07 to 0.54×10⁸ M ^-1 for HSA and from 2.16 to 0.87×10⁷ M ^-1 for BSA.
These data indicate that the histidyl residues are relatively unimportant for maintaining the native tertiary structure of BSA and HSA, but they are critical for determining the binding capacity of the albumins. Our data also imply that the tertiary structure of the BSA molecule is more labile than that of HSA.     

Electrometric, spectroscopic and thermogravimetric studies on lanthanide-arylhydrazones of benzoylacetone and dibenzoylmethane complexes

The chelate formation reaction of La^III, Pr^III, Nd^III, Gd^III, Ho^III and Er^III ions with some arylhydrazones of benzoylacetone (BAAH) and dibenzoylmethane (DBMAH) has been investigated by conductimetric and pH-metric titration. The chelates formed have 1:1 and 1:2 stoichiometries (M³⁺: L). The stability constants of the metal chelates are correlated to the atomic numbers Z, the ionic radii r, the ionization potentials and the electronegativities of the lanthanide ions.

The solid complexes have been isolated and their structures have been assigned on the basis of elemental analysis, IR and NMR spectroscopy and thermogravimetric analysis (TG). It has been determined that the chelation occurs through the -NH group and one carbonyl group of the β-diketone, in addition to the OH⁻ group. The coordination number ranges from six to eight according to the number of coordinated water molecules which are confirmed by thermogravimetric analysis. The structural formulae are suggested.     

PHOTOLYSIS OF MELANIN PRECURSORS: FORMATION OF SEMIQUINONE RADICALS AND THEIR COMPLEXATION WITH DIAMAGNETIC METAL IONS

Abstract— Semiquinone free radicals have been generated by the UV photolysis of aqueous solutions of melanin precursors (catechols and catecholamines) and characterized by electron spin resonance (ESR) spectroscopy. Hyperfine parameters of the semiquinones are sensitive to the nature of the substituents on the aromatic ring, reflecting (i) the ionization state of the substituent; (ii) restricted rotation of methylene protons; and (iii) the presence of a chiral carbon center. In the presence of complexing metal ions (Zn²⁺, Cd²⁺) the semiquinones form chelate complexes whose ESR spectra differ from those of the uncomplexed radicals. Satellite peaks demonstrating hyperfine coupling to metal isotopes (⁶⁷Zn, ¹¹¹Cd and ¹¹³Cd) present in natural abundance were detected in several instances. Concentrations of metal complexes are much higher than those of the uncomplexed radicals, suggesting that diamagnetic metal ions may be useful in identifying the presence of semiquinone radicals of this kind in various systems.     

EFFECT OF METAL IONS ON THE LUMINESCENCE OF ACRIDINE DYES BOUND TO DNA

Abstract— The luminescence of acridine dyes intercalated in DNA was studied as a function of the concurrent binding of metal ions to DNA, in an effort to deduce specific site interactions of the dyes. Two dyes, proflavine (PF) and acridine orange (AO), and two metal ions, silver and mercuric, were used. Both ions quench the fluorescence of the dyes in aqueous solution at room temperature. The metal ions have a different effect on the fluorescence of these dyes when they are intercalated between the base pairs of DNA. The fluorescence of AO is decreased when silver is bound, while the fluorescence of PF is enhanced. Since Ag⁺ initially binds to GC sites in DNA, which quench the PF fluorescence, it ostensibly 'turns off' the quenching by DNA at these sites, and this effect is greater than the quenching effect of the silver ion itself. Hg²⁺ ion initially binds to AT sites in DNA. Since both dyes fluoresce from AT sites, Hg²⁺ is expected to quench their fluorescence. This behavior is observed at low r (metal ion/base). At higher r values, however, where Hg²⁺ is expected to begin binding to GC sites, the fluorescence of PF is enhanced. These quenching turn-off effects are tentatively interpreted in terms of a change in the structure of the dye/DNA complex which occurs when a metal ion binds at the intercalation site. At 77 K. no fluorescence enhancement is observed when metal ions bind; Ag⁺ quenches the fluorescence and enhances the phosphorescence of both dyes. Qualitatively similar results are obtained with Hg²⁺.     

ULTRAVIOLET IRRADIATION OF NUCLEIC ACIDS COMPLEXED WITH HEAVY ATOMS—III. INFLUENCE OF Ag + AND Hg 2+ ON THE SENSITIVITY OF PHAGE AND OF TRANSFORMING DNA TO ULTRAVIOLET RADIATION

Abstract— We have studied the influence of the heavy metal ions Ag⁺ and Hg²⁺ on the photoinactivation and photodimerization of transforming DNA and of bacteriophage. The rate of inactivation of Haemophilus influenzae transforming DNA by ultraviolet (UV) radiation was enhanced by a factor of 30 when it was complexed with Ag⁺. This enhancement was correlated with a comparable increase in the rate of thymine dimerization. In contrast, mercuric ions led to a reduction in the rates of both inactivation and dimerization. When we examined the effects of these metal ions on the photobiology of bacteriophage, we again found that Ag⁺ enhanced and Hg²⁺ reduced the rate of ultraviolet inactivation. These results demonstrate that heavy metals may be useful tools for studying the photochemistry and photobiology of nucleic acids.     

MIGRATION OF EXCITATION ENERGY FROM THIONINE TO METHYLENE BLUE IN MICELLES

Abstract— The main absorption bands of thionine (Th⁺) and methylene blue (MB⁺) in aqueous solution lie at 598 nm and 664 nm, respectively. This position permits excitation energy transfer from Th⁺ to MB⁺, but not vice versa. We describe here studies of such transfer between these molecules adsorbed on micelles of sodium lauryl sulfate (SLS), imitating, at least to some extent, the state of pigments in chloroplasts.
The SLS concentration was varied from 3.0 to 11 × 10^-3 M. In the presence of dye, aggregation to micelles, each containing 70–100 detergent molecules, begins at about 3.0 × 10^-3 M SLS. Practically all dye ions are adsorbed on these micelles as soon as their formation begins.
Energy transfer from adsorbed Th⁺ ions to adsorbed MB⁺ ions can be demonstrated by observing the quenching of the fluorescence of thionine and the sensitization of that of methylene blue.
At [Th⁺] = [MB⁺] = 1 × 10^-5 M , the most efficient energy transfer (82 per cent efficiency, as derived from measurements of the quenching of Th⁺ fluorescence, or 90 per cent, as derived from sensitization of MB⁺ fluorescence) is observed at the lowest SLS-concentration (3.0 × 10^-3 M ), when the only micelles present are those formed by aggregation of dye-carrying low molecular complexes of SLS with dye cations. Each micelle carries, under these conditions, 10–14 molecules of the two dyes, and the distance between two closest dye ions is about 16 A. Transfer becomes less efficient as the SLS-concentration increases, causing pigment molecules to distribute themselves among a greater number of micelles.     

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.     

PHOTODYNAMIC INACTIVATION OF LYSOZYME BY EOSIN

Abstract— It has been demonstrated that singlet oxygen is the major oxidizing entity in the photo-dynamic inactivation of hen egg white lysozyme by eosin, using D₂O to enhance the solvent-induced decay lifetime, and azide ion as a specific scavenger. Two regimes of inactivation can be distinguished depending on whether the sensitizer is free or complexed to the enzyme. The kinetic analysis for free dye sensitization, based on photostationary measurements and inactivation quantum yields, indicates that at least 1 in 15 singlet oxygen interactions with lysozyme leads to loss of lytic activity. The direct attack of triplet eosin makes a lesser overall contribution in air-saturated solutions, where 1 in 4 reactions induces inactivation. Lysozyme binds 1 eosin molecule from pH 4 to 12, leading to almost total quenching of the tryptophyl residue fluorescence without inhibition of the enzymic activity. The inactivation quantum yields indicate that singlet oxygen generated from the bound dye is the inactivating agent, but the dominant attack takes place with the complexed fraction of lysozyme molecules. The tryptophyl residue loss is the same or smaller in changing from H₂O to D₂O despite the 5–10 times increase in quantum yield, indicating that singlet oxygen inactivates also by reacting with residues other than tryptophan. The photochemical and fluorescence results are consistent with the the identification of tryptophyl site 108 with the eosin binding site and a reaction target for singlet oxygen. In a re-examination of earlier work on eosin-sensitized photo-oxidation of I", it has been found that singlet oxygen is the oxidizing agent in aerobic solutions.     

Self-assembly of lanthanide mixed-carboxylates coordination polymers

Two new mixed-ligands lanthanide coordination polymers, [Ln(Ac)(ip)(H₂O)₂]·0.5H₂O (Ln=La (1); Ln=Eu (2); Ac=acetate; ip=isophthalate) have been synthesized under hydrothermal condition. Single-crystal X-ray analyses show that complexes 1 and 2 are three-dimensional structure in which lanthanide ions are bridged by monocarboxylate ligand, acetate or dicarboxylate ligand, isophthalate. And the central lanthanide ions, La³⁺ and Eu³⁺, are both nine-coordinate with oxygen atoms. The thermogravimetric analysis was carried out to examine the thermal stability of the title complexes. And the photoluminescence property of complex 2 was also investigated.     

HEMATOPORPHYRIN PHOTOSENSITIZATION OF SERUM ALBUMIN and SUBTILISIN BPN'

—The photosensitized inactivation of subtilisin BPN' by free hematoporphyrin (HP) followed exponential kinetics with positive mechanistic tests for the involvement of singlet oxygen (¹O₂) as the principal intermediate. The photoinactivation quantum yield was 0.029 at 390 nm in oxygen-saturated, D₂O buffer at pH 7.0. The effects of HP binding were investigated for tryptophan oxidation in bovine serum albumin (BSA) and human serum albumin (HSA) at high protein:HP concentration ratios where the HP was > 97% complexed. The reaction kinetics were non-exponential and mimick a second-order process in the initial stages. The rate of HP photobleaching was 30-fold faster for complexed HP compared with free HP, which was shown to account for the observed kinetics. Mechanistic tests showed that ¹O₂ was the dominant photooxidizing intermediate of tryptophan residues and that it was not involved in the accompanying photobleaching of HP. The quantum yield for tryptophan oxidation in BSA was 0.11 at 390 nm in oxygen-saturated, D₂O buffer at pH 8.0. The reactivity of HSA was approximately 2-fold lower than BSA for equivalent conditions. Estimates of the reaction cross sections led to 3 Ų for the inactivation of subtilisin BPN' by ¹O₂ and 20 Ų for the oxidation of tryptophan in BSA.     

络合物稳定常数的计算

本文介绍了计算络合物稳定常数的八种方法,它们是:1.lgK₁=Az²/r+Bx_±+C.2.两相似金属离子与一系列配位体络合时,lgK₁间一的线性关系。3.两相似配位体与一系列金属离子络合时,lgK₁间的线性关系。4.一系列相似配位体与同一金属离子络合时,lgK₁与∑pK_a间的线性关系。5.一系列相近z²/r的金属离子与同一配位体络合时lgK₁与x_±间的线性关系。6.一系列相近x的金属离子与同一配位体络合时lgK₁与z²/r间的线性关系。7.金属离子随z²/r和x而变化的络合次序。8.∑pK_a较大的配位体与z²/r较大的金属离子匹配,电负性较小的配位体与电负性大的金属离子匹配。     

SYNTHESES AND SPECTROSCOPIC PROPERTIES OF LANTHANIDE COMPLEXES, Ln(acac)_3(Ph_3PO)_3

A series of new complexes of formula Ln(acac)_s(Ph_3PO)_3 (where Ln~(3+)=Nd~(3+), Sm~(3+), Eu~(3+), Tb~(3+), Ho~(3+)) have been prepared and characterized. The coordination of two ligands with lanthanide ions are studied by IR spectra, ~1H, ~(13)C, ~(31)P-NMR and metal-ligand charge tranafer tranaition is also discussed via electronic adaorption and luminescence spectroscopy.     

SINGLET ENERGY TRANSFER BETWEEN AROMATIC AMINO ACIDS AND NUCLEIC ACID BASES. THEORETICAL CALCULATIONS

Abstract— Critical Forster distances for excitation energy transfer at the singlet level between the tyrosyl or tryptophyl residues of proteins and nucleic acid bases have been calculated. Efficient singlet energy transfer can be expected from tyrosine to nucleic acid bases both at room temperature (10 0 < 17 A) and at low temperature (20 < R₀ < 23 Å). At low temperature nucleic acid bases should be able to transfer their singlet excitation energy to the indole ring of tryptophan with a reasonable probability (9 < R₀ < 15 A).     

PHTHALOCYANINE PHOTOSENSITIZATION OF MAMMALIAN CELLS: BIOCHEMICAL and ULTRASTRUCTURAL EFFECTS

Abstract— The concentrations of Na⁺, K⁺, Ca²⁺, Mg²⁺ and CI⁻ ions in the cytoplasm of octopus photoreceptor cells were determined before and after illumination by electron probe X-ray microanalysis. The concentrations of these elements in the dark-adapted photoreceptor cells were: Na⁺, 68.4; K⁺, 111.4; Ca²⁺, 4.0; Mg²⁺, 16.4; and CI⁻, 102.9 m M /kg of cytoplasm. Illumination raised the concentration of Na⁺ by 58 m M and that of Cl⁻ by 23 m M and reduced the K⁺ concentration by 47 m M /kg of cytoplasm. A trace increase of intracellular Ca²⁺ and a trace decrease of Mg²⁺ were observed. These results confirm the hypothesis that sodium ions flow in on illumination, and suggest the influx of chloride ions and the outflux of potassium ions during illumination. The intracellular concentrations of Na⁺, K⁺ and Cl⁺ can give the basis for calculating the ion permeability of ion channels in octopus photoreceptor cell membranes, using values of the membrane potentials obtained by electrophysiological studies     

EFFECT OF TEMPERATURE AND pH ON THE QUENCHING OF TRIPLET LUMIFLAVIN IN THE PRESENCE AND ABSENCE OF FERRI- AND FERROCYANIDE

Abstract— –Flash photolysis at 450 nm over the temperature range 0.8–60°C was used to determine Arrhenius parameters for the first and second order disappearance of triplet lumiflavin (1.66 µ .M ) at a flash energy of 2 kj in deaerated phosphate buffer at varying pH:
³Lf → Lf₀
³Lf +³Lf → Lf₀+ Lf₀
Arrhenius parameters were also determined for the pseudo first-order quenching of triplet lumiflavin by 10 µ M ferri- and ferrocyanide ions,
³Lf + Fe³⁺→ Fe³⁺→ Lf₀+ Fe³⁺ (energy transfer)
³Lf + Fe²⁺→ Lf^-+ Fe³⁺ (electron transfer)
and for disappearance of the semireduced lumiflavin in the presence of ferrocyanide at pH 6.8, by the second-order reaction
Lf^-+Lf ^-→ Lf₀+ Lf⁼.     

EFFECT OF PARAMAGNETIC SPECIES ON 8-METHOXYPSORALEN-SENSITIZED PHOTOINACTIVATION OF BACTERIOPHAGE LAMBDA

Abstract. 8-Methoxypsoralen-sensitized photoinactivation of bacteriophage lambda (Λ_vir) was examined in the presence of various metallic ions. Paramagnetic ions, Co²⁺, Ni²⁺ or Mn²⁺, strongly repressed the reaction, whereas diamagnetic ions, Ca²⁺ or Zn²⁺, exerted no influence. Significant repression was also observed in the sample solution equilibrated with air.