癌症病人血液的荧光光谱研究

研究了癌症病人全血、血浆和血红蛋白的激发波长—发射波长—荧光光强三维光谱,观察到特征谱带625nm的荧光本原物质存在于血红蛋白部分,比较了十五种癌症(28例),十例非癌症病人和健康人血液在625nm带的荧光强度.     

发菜藻胆体分离和光谱特性的研究

完整藻胆体和不完整藻胆体的吸收峰都在618nm。完整藻胆体的室温荧光峰位于670nm 以上,而不完整藻胆体则在670nm以下。完整藻胆体的77K荧光发射光谱中只有648nm一个荧光发射带;而在不完整藻胆体,则有2个或3个发射带,它们位于684nm,666nm和648nm, 依次属于别藻蓝蛋白 — B,别藻蓝蛋白和C — 藻蓝蛋白的荧光。     

测定植物胞内游离钠离子的研究进展

植物耐盐机制的研究一直是植物抗性研究的焦点。近年来,随着生物学不断发展和新荧光标记技术的运用,胞内钠离子测定逐渐应用于植物盐胁迫研究中。该文论述了以下三方面问题:(1)分别介绍了SBFI、Sodium Green和Coro Na Green三种钠离子荧光指示剂:SBFI是一种双激发波长指示剂,其激发波长是340 nm/380 nm,发射波长是500 nm;Sodium Green和Coro Na Green是单波长指示剂,其激发波长分别是507 nm和492nm,发射波长分别是532 nm和516 nm;(2)比较了酯导入、酸导入、电穿孔和显微注射等几种常见荧光指示剂载入胞内方法的优缺点,重点介绍了一种无损伤低温抑制酯酶法:先将荧光指示剂在缓冲液中低温(4℃)处理2 h,随后回到常温(20℃)在不含荧光指示剂的缓冲液中孵育2 h;(3)阐述了胞内离子浓度计算公式,包括单波长测定公式、双波长比率测定公式。     

血卟啉衍生物——组蛋白系统照光后光动力学效应的观察:一种新荧光物质的生成

血卟啉衍生物(Hpd)和组蛋白经照光产生一种新荧光物质.其发射波长为460nm,激发波长为370nm.当照光在43℃高温情况下进行时,高温对Hpd的光动力学效应有协同作用.用牛血清白蛋白、血红蛋白,核糖核酸酶、胰蛋白酶及培养的中国仓鼠细胞代替组蛋白均能生成新荧光物质.而所测试的半胱氨酸、酪氨酸、苯丙氨酸、赖氨酸及组氨酸中.唯有组氨酸可产生相似的荧光物质.这种荧光物质推测是Hpd与组蛋白中组氨酸所形成的一种加成物.形成新荧光物质的光化学反应机制主要是单线态氧(~1O_2)的氧化作用.     

不同激发波长的人体血清自体荧光光谱分析

利用分子荧光光度计测定了激发波长分别为457.9 nm,476.5 nm,488.0 nm,501.7 nm和514.5 nm光的血清自体荧光光谱,并对这些光谱的特征和产生机制进行了分析。实验和分析结果表明:在500 nm~750 nm的波长范围内,518 nm和640 nm附近有两个比较明显的荧光峰;590 nm处有一个非常弱的峰。它们可能主要来自于血清中胆红素,核黄素及其衍生物,β-胡萝卜素,锌卟啉及原卟啉IX等的贡献。这些研究结果将为利用氩离子激光器作为光源进行光谱诊断研究选择最佳激发波长提供一些参考。     

雨生红球藻(Haematococcus pluvialis)的光谱特性研究

实验测定了雨生红球藻不同生长阶段的色素组成,吸收光谱,荧光光谱,并对其进行了分析。结果表明,用490nm波长激发时,雨生红球藻在绿色细胞阶段存在710nm和730nm附近的长波长荧光发射峰,而在红色细胞阶段仅存在730nm的长波长荧光发射峰,预示着雨生红球藻不同生长阶段在PSⅠ结构,组成,及其色素蛋白的排布等方面有很大差异。     

紫光激发中药光敏剂用于早期胃癌的诊断

采用405nm紫光激发传统中药光敏剂(CpD4)发射的荧光中心波长在660nm。红色荧光能深入组织,因而能够应用在胃癌早期诊断及治疗中。本文测定了中药光敏剂的吸收光谱和发射荧光光谱,并提出了二种用于激发光敏剂的紫光光源。一种为“Hg-Xe”灯,发射峰为433nm;另一种为采用紫光LD,发射峰为405nm。这二种波长和中药光敏剂的吸收峰完全匹配。     

基于微流控芯片多波长荧光检测体系

利用四个LED分别匹配相应的激发滤光片,带通发射滤光片和PMT,自行搭建微芯片多波长荧光检测系统。用于复杂生物混合样品:四类试样(荧光胺标记的牛磺酸(FT Ex/Em 390/446nm)、荧光素(Fl Ex/Em 480/520nm)、5(6)-羧基-X-罗丹明(ROX Ex/Em 563/600nm)和花菁染料(Cy 5 Ex/Em 635/677nm)的同时分离和测定并得到充分的验证。     

固氮蓝藻Anabaena sp.7120的叶绿素蛋白复合体的分离和光谱特性的研究

用光合膜片增溶和SDS-聚丙烯酰胺凝胶电泳方法,从固氮蓝藻Anabaena sp.7120分离到7条色素带。迁移率较慢的五条叶绿素蛋白复合体带,具有相同的吸收光谱和室温荧光光谱特性。它们的红区最大吸收峰在676nm;蓝区最大吸收峰在438nm。它们的室温荧光发射最高峰在672—673nm;在710,732和740nm都有小峰。这些是CPⅠ叶绿素所特有的。我们认为这5条带都是属于光系统Ⅰ的叶绿素蛋白复合体。另一条迁移率稍快的叶绿素蛋白复合体带为CPⅡ。它的红区最大吸收峰在672nm;蓝区最大吸收峰在436nm。与CPⅠ带相比,两个峰均向短波端偏移。它们的室温荧光发射最高峰在675nm,没有CPⅠ所特有的小峰。这些性质说明此带和CPⅠ带不同,而是和光系统Ⅱ反应中心相关的一个复合体。迁移率最快的带是游离色素带。     

荧光分光光度法测定谷物蛋白质中的酪氨酸

本文探讨了谷物中酪氨酸的荧光测定法。将30毫克风干样置玻璃水解管中,以6Mol盐酸作水解剂,110℃真空水解22小时,水解产物减压脱酸,以去离子双蒸水溶解,在激发波长277nm,发射波长303nm处测其荧光强度。方法精确度良好,重复测定12次变异系数为0.98％。在5ng～2μg/ml标准酪氨酸范围内,回收率为96—105％。用此法测得谷物中酪氨酸与氨基酸分析仪测得值相符。     

A comparison of the heme electronic states in equilibrium and nonequilibrium protein conformations of high-spin ferrous hemoproteins. Low temperature magnetic circular dichroism studies

The visible and near infrared magnetic circular dichroism (MCD) spectra of equilibrium high-spin ferrous derivatives of myoglobin, hemoglobin, horseradish peroxidase and mitochondrial cytochrome c oxidase at 15 K are compared with those of the corresponding proteins in nonequilibrium conformations produced by low-temperature photodissociation of CO-complexes of these proteins as well as of O2-complexes of myoglobin and hemoglobin. Over all the spectral region (450-800 nm) the intensities of MCD bands of hemoproteins studied in equilibrium conformation are shown to be strongly temperature-dependent, including a negative band at ca. 630 nm and positive bands at ca. 690 nm and at ca. 760 nm. In contrast to the absorption spectra, the low-temperature MCD spectra of high-spin ferrous hemoproteins differ significantly, reflecting the peculiarities in the heme iron coordination sphere which are created by a protein conformation. The MCD spectra reveal clearly the structural changes in the heme environment which occur on ligand binding. On the basis of assignment of d leads to d and charge-transfer transitions in the near infrared region the correlation is suggested between the wavelength position of the MCD band at approx. 690 nm and the value of iron out-of-plane displacement as well as between the location of the band at approx. 760 nm and the Fe-N epsilon (proximal histidine) bond strength (length) in equilibrium and nonequilibrium conformations of the hemoproteins studied. The high sensitivity of low-temperature MCD spectra to geometry at heme iron is discussed.     

Near-infrared spectra of Scapharca homodimeric hemoglobin: characterization of the deoxy and photodissociated derivatives.

The near-infrared charge transfer band at 760 nm (band III) has been investigated in deoxy and photodissociated dimeric Scapharca hemoglobin. At 300 K, the 10-ns spectrum of the carbonmonoxy derivative photoproduct is shifted by about 6 nm toward longer wavelengths with respect to the deoxy spectrum, both in buffer and in glycerol/buffer solutions. Moreover, the band III peak occurs at about the same wavelength at 300 K and at 10 K for the 10-ns photodissociated derivative, whereas in the deoxy derivative large changes in peak position and linewidth are observed as a function of temperature. These findings suggest that in dimeric Scapharca hemoglobin the photoproduct has not relaxed after 10 ns. The complete time dependence of the relaxation process has been studied both in buffer and in glycerol/buffer solutions at room temperature. The relaxation from the photoproduct to the deoxy species occurs on a microsecond time scale, in line with recent optical absorption and resonance Raman measurements.     

Effects of Mg2+ and Ca2+ on photoinduced Euglena flagellar responses

The flagellar frequency and waveform of Euglena were analyzed under full illumination (420-700 nm) and in a restricted wavelength band (530- 700 nm) when the cells were in a medium containing Mg2+ or had been microinjected with Mg2+, Mn2+, or Ca2+ in solution. Magnesium abolished the change in flagellar frequency and the reversal in waveform that cells exhibit when illuminated by a 530-700 nm wavelength band. Under this restricted illumination, Ca2+ caused an increase in flagellar waveform reversal and a decrease in beating frequency. The flagellar motility of cells impaled on a microelectrode was examined in cells illuminated with various wavelengths.     

Solution studies on heme proteins. Circular dichroism and optical rotation of Glycera dibranchiata hemoglobins

Circular dichroism (CD) and optical rotatory dispersion (ORD) spectra of several liganded derivatives of the monomer and polymer hemoglobin components of the marine annelid, Glycera dibranchiata were measured over the wavelength range 650--195 nm. The differences observed between the monomer and polymer components for the heme dichroic bands in the visible, Soret and ultraviolet wavelength regions seem to result from changes in the heme environment, geometry and coordination state of the central heme iron in these proteins. Within the Soret region, the liganded derivatives of the monomer hemoglobin exhibit predominantly negative circular dichroic bands. The heme band at 260 nm is also absent for the monomer hemoglobin. The ORD and CD spectra in the far-ultraviolet, peptide absorbing region suggest also differences in the alpha-helix content of the monomer and polymer hemoglobins. The values for the single-chain G. dibranchiata hemoglobin are in the expected range (about 70% alpha-helix) as predicted by the X-ray structure of this protein. The lower estimates of the alpha-helix content for the polymer hemoglobin (approx. 50%), may reflect the differences in amino acid composition, primary structure and polypeptide chain foldings. Changes in oxidation state and ligand binding appears to have no pronounced effect on the helicity of either the monomer or polymer hemoglobins. The removal of the heme moiety from the monomer hemoglobin did result in a major decrease in its helix content similar to the loss of heme from myoglobin.     

The detection of cytochrome oxidase heme iron and copper absorption in the blood-perfused and blood-free brain in normoxia and hypoxia.

The resolution of cytochrome and hemoglobin changes in in vivo rat and cat brains has required studies over wide wavelength ranges (580-1100 nm) with a novel spectroscopic technique using blood-free and blood-perfused brains. Tissue oxygen was varied from physiological levels to 0 and hematocrits were varied from normal to less than 1%. The experimental results were subjected to a multicomponent analysis using the Beer-Lambert law. At normal hematocrits, the oxygen saturation of hemoglobin in the brain was found to be 30-50% in rats and cats, indicating that the optical method responded primarily to the saturation of the venous ends of the capillary beds. With low hematocrits, both brains showed the absorption band of reduced cytochrome c, the iron component of cytochrome aa3, plus the absorption band of the oxidized copper component. In cat brains, the background absorption changed at all wavelengths. Thus, no isosbestic points were observed in the spectra. In rat brains, however, they were readily observed. The "overtones" of water absorption in the NIR region were found to be significant in the difference spectra of the cat brain, but not in the rat brain. Parallel absorbance changes in the heme and copper components of cytochrome aa3 were obtained in rat and cat brains during the normoxic-hypoxic transition. The ratio of the iron absorbance at 605 nm to the copper absorbance at 830 nm is much smaller in both brains than the in vitro value due to the shorter path length of photon migration at the shorter wavelengths.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vitamin B6 fluorescence in cells]

The band of cell fluorescence with the maximum of 395-400 nm is registered. This band is exposed on the phone of the tryptophane by wavelength excitation lambdaex=250-260 nm, and in pure scape by lambdaex=310-326 nm. Pyridoxin - substrate of vitamin B6 has identical parameters of spectra excitation and emission of neutral (pH 7) and acid (pH 2) solutions. After temperature damage of cells the intensity of this band increases.     

Studies on the charge transfer band in high spin state of ferric myoglobin and hemoglobin by low temperature optical and magnetic circular dichroism spectroscopy.

The behavior of charge transfer band, appearing at 600-650 nm in ferric high spin derivatives of myoglobin and hemoglobin, was studied under various conditions by low temperature optical and magnetic circular dichroism spectroscopy. Optical absorption spectra have demonstrated that: (1) The charge transfer band at 630 nm of myoglobin (Fe3+)-H2O (pH 7.0) at room temperature split into three bands, 627 nm, 645 nm and 664 nm (shoulder) at 77 degrees K, whereas that of hemoglobin (Fe3+)-H2O showed no splitting. (2) By lowering the pH value from 7.5 to 4.3 this splitting in myoglobin was observed to disappear only in the presence of a small amount of phosphate ion, accompanying a midpoint at pH 6.7 +/- 0.1. This does not originate from the released hemin. (3) Hemin (pH 7.55) showed no splitting of the charge transfer band at 77 degrees K. (4) This splitting depended on the species of 6th ligand. For myoglobin-F- the splitting could scarcely be observed, whereas the proton-donating ligands such as HCOOH and CH3OH exhibit the splitting as well as H2O. Magnetic circular dichroism spectra have demonstrated that: (5) The charge transfer band at 600-500 nm indicated Faraday A term and B term. (6) A negative B term band was observed at 650 nm for myoglobin-H2O in the glassic solvent of potassium glycerophosphate-glycerol, whereas it was not observed for hemoglobin-H2O. Several discussions were performed on the origin of splitting of the charge transfer band in myoglobin-H2O. It is now concluded that the hydrogen bond between the 6th ligand and the distal histidine contributes to the splitting of the charge transfer band around 630 nm for myoglobin Fe3+)-H2O at low temperature and that disappearance of the splitting at low pH is originated from the presence of phosphate ion.     

Spirocamallanus cricotus (Nematoda): isoelectric focusing and spectrophotometric characterization of its hemoglobin and that of its piscine host, Micropogonias undulatus

The hemoglobins of Spirocamallanus cricotus, a reddish-colored, camallanid nematode, and its Atlantic croacker fish host, Micropogonias undulatus, were characterized with spectrophotometry and isoelectric focusing. Hemoglobin from female parasites' perienteric fluid and homogenized male parasites gave Spectrophotometric peaks at 412, 539, and 575 nm, whereas female worms drained of perienteric fluid and homogenized differed by having a Soret peak of 408 nm. Changing the ionic strength of the buffer from 0.1 to 0.01 M shifted the Soret peak to 406 nm for the female parasites' perienteric fluid and ground male parasites and 404 nm for homogenized female parasites. In all cases, the β band had a higher absorption than the α band suggesting a high O₂ affinity for the parasite hemoglobin. Host hemoglobin had peaks of 406, 437, and 577 nm. Isoelectric focusing not only confirmed the Spectrophotometric evidence that host and parasite hemoglobins differed, but also showed that the parasite's analyzed hemoglobin fractions differed from one another by having different isoelectric points.     

The optical spectra of fluoride complexes can effectively probe H-bonding interactions in the distal cavity of heme proteins

Fluoride complexes of heme proteins are characterized by unique spectroscopic properties, that provide a simple and direct means to monitor the interactions of the distal heme pocket environment with the iron-bound ligand. In particular, a strong correlation has been demonstrated between the wavelength of the iron-porphyrin charge transfer band at 600-620 nm (CT1) and the strength of H-bonding donation from the distal amino acid side chains to the fluoride ion. In parallel, resonance Raman spectra with excitation within either the CT1 band or the charge transfer band at 450-460 nm (CT2) have revealed that the iron-fluoride stretching frequency is directly affected by H-bonding to the fluoride ion. On this basis, globins and peroxidases display distinct spectroscopic features, which are strongly dependent on the capability of their distal residues (i.e. histidine, arginine and tryptophan) to be involved in H-bonding with the ligand. In particular, in peroxidases strong H-bonding corresponds to a low iron-fluoride stretching frequency and to a red-shifted CT1 band. The reverse is observed in myoglobin. Interestingly, a truncated hemoglobin of microbial origin (Thermobifida fusca) investigated in the present work, displays the specific spectroscopic signature of a peroxidase, in agreement with the presence of strong H-bonding residues, i.e., tyrosine and tryptophan, within the distal pocket.