Light-induced spectral changes of carotenoids in chromatophores of Rhodopseudomonas sphaeroides

Carotenoid difference spectra were recorded of chromatophores of Rhodopseudomonas sphaeroides energized with low light intensities versus chromatophores under dark conditions. The amplitudes of the peaks and troughs were dependent on the light intensities and the duration of illumination, but the shape of the difference spectra remained constant. Sharp isosbestic points were found at 515, 500, 481, 465, and 452 mm. When potassium-valinomycin-induced diffusion potentials (inside negative) were imposed on the chromatophores “mirror images” of the light-induced difference spectra were recorded with the same isosbestic points and the same positions but different relative amplitudes of the peaks and troughs. The results are explained in terms of changes in the shape of the vibrational splittings of the carotenoid main electronic transition. This could be the result of changes in the fluidity of the environment of the carotenoids upon energization of the membrane. Prolonged periods of illumination with low or high light intensities resulted in irreversible changes of the difference spectra. Short periods of illumination with high light intensities resulted in reversible elevations of the baseline and red shifts of peaks, troughs, and baseline crossings.     

Geographical Variation in the Characteristics of Absorption Spectra and Fluorescence Spectra of Pinus armandi

By measuring the derivative absorption spectra of chloroplasts of different populations of Pinus armandi Franch., it was found that southern populations maintained higher absorption at 680 nm than at 670 nm, but some of the northern populations deviated the maximum absorption from 680 nm to 670 nm, which indicated that the activity of PS Ⅱ in some of the northern populations declined. Clear geographical differences also have been found in the positions of emission peaks of PS Ⅰ and PS Ⅱ in the fluorescence emission spectra at 77 K. Analysis of the fluorescence excitation spectra at 77 K revealed geographical changes in the absorption.status of Chl a. Besides, the experimental results indicated that the intact needles of Pinus armandi are not ideal materials to be used in detecting the geographical variation in photochemical reaction process because the presence of thicker coat, resin etc. can conceal the spectral differences in different populations.     

华山松吸收光谱及叶绿素荧光特性的地理变异

利用分离的叶绿体作实验材料,发现华山松(Pinus armandi Franch.)南方种源的4阶导数吸收光谱在680nm处峰值较大,在670nm处峰值较小,而北方种源中出现了在680nm处峰值较在670nm峰值小的类群,推断北方种群反应中心活力有下降趋势。南、北种源之间的低温(77K)荧光发射光谱有明显差异,PSⅠ、PSⅡ发射峰位置出现地理变动。低温荧光激发光谱分析表明,地理变异主要发生在叶绿素a的分子状态上。研究还表明,完整的针叶因为有角质层、松脂等物质干扰,检测不出光谱的差异,不是理想的实验材料。     

Changes of the antenna of photosystem I induced by short-term heating

Changes in low-temperature fluorescence spectra of pea chloroplasts induced by the short-term heating were studied. Excitation spectra of the long-wavelength fluorescence were studied as well. Heating was carried out at 45°C for 5 min in the darkness or in the presence of white light sourced with intensities of 260 or 1400 μmol/m² s. All variants of heating decreased the intensity of the long-wavelength fluorescence band. The integral of the excitation spectrum decreased after the exposure to heating in the darkness and increased after the exposure to heating in the presence of light. The observed changes in most intensive components — 726, 729 and 731 nm — of the long-wavelength fluorescence band, induced by various modes of heating, were similar. The changes in the fourth intensive component at 735 nm were different. Twenty-five components were found in the fine structure of the excitation spectrum of the long-wavelength fluorescence. Positions of most of peaks corresponded to the absorption peaks of Lhca proteins. Heat-induced changes in the excitation spectrum in the regions corresponding to the absorption of chl b and short-wavelength forms of chl a have been shown to correlate with changes in the intensities of the 726-, 729-, and 731-nm components of the long-wavelength fluorescence. This allows one to assign them to the emission of the outer antenna of Photosystem I. Changes in the intensity of the component at 735 nm correlated only with changes in excitation spectrum in the long-wavelength region that corresponded to the absorption of the long-wave-length forms of chlorophyll a. Therefore, the 735-nm component could be assigned to the emission of the Photosystem I inner antenna. Analysis of the changes induced by heating in the emission and excitation spectra of fluorescence revealed changes in the energy transfer in the outer and the inner antennas of Photosystem I. Heating in the darkness lowered the energy transfer in the outer and in the inner antennas. Both modes of heating in the presence of light increased the energy transfer in the outer antenna. For the inner antenna, presence of the light promotes an efficient of energy transfer at the levels close to the control one. It is proposed that illumination during heating exposure causes a specific state of the antenna complex in Photosystem I that provides an increase in funneling of the energy toward the reaction centers.     

Identification Mutants of Plant Pigment with Spectral Technology

Differences in the pigment and thylakoid membranes levels among the mutant barley 1832C, Chlorina-f2 was compared with a normal one by means of their absorption spectra and the 4th derivative spectra. Results showed that there was no absorption peaks for Chl b at 651 nm in red and 470 nm in blue regions in the absorption and the 4th derivative spectra in the mutant thylakoid membranes. At the same time there was no absorption peaks of Chl b at 645 nm and 455 nm in the absorption and the 4th derivative spectra in its acetone (80%) extract. Therefore, mutant barley 1832C was proved to be a new type of chlorophyll b-less mutant that could survive independently in nature.     

Heme-linked spectral changes of the protein moiety of hemoproteins in the near ultraviolet region

Spectral changes of hemoproteins in the near ultraviolet region on binding to a ligand and on oxidation-reduction of the heme-iron were studied by computer-controlled spectrophotometry. Near ultraviolet difference spectra between the low spin and high spin forms of ferric hemoproteins were classified into three groups: Those showing two absorption peaks having maxima at around 285 and 295 nm, those showing a peak at around 275 nm, and those showing a peak at around 300 nm. No corresponding absorption peak was observed with model heme complexes of low molecular weight. The intensity of the peak in cyanide difference spectra of catalase and horseradish peroxidase in the near ultraviolet region was dependent on the concentration of added cyanide and paralleled the intensity of the spectral changes in the Soret region. The spectral changes in both the near ultraviolet and Soret regions developed within 6 ms after the addition of cyanide. Difference spectra between the reduced and oxidized forms of cytochrome c, cytochrome oxidase-cyanide complex, hemoglobin, and lactoperoxidase-cyanide complex showed a characteristic peak at around 285-290 nm. Various difference spectra of hemoglobin in the near ultraviolet region were also measured. The observed positions, shapes, combinations, and relative intensities of the peaks were compared with those of solvent perturbation difference spectra and pH difference spectra of proteins and aromatic amino acids and also with the diacetylchitobiose-induced difference spectrum of lysozyme. The kinds of aromatic amino acid residues possibly responsible for the observed difference peaks were discussed on the basis of the results of the comparison. Based on the results obtained, the common occurrence of a heme-linked functional response of the hemoprotein conformation was suggested.     

用光谱技术鉴定植物色素突变体

1983年我国报道了从γ-射线处理的“矮杆齐”大麦中得到了一株黄绿色的突变体1832C[1]。本文用光谱技术对该突变体的光合色素成分进行了鉴定。1　材料和方法　　材料为六棱裸大麦“矮杆齐”和由该品种大麦诱变形成的黄绿色突变体1832C(Mb1832C),以及作为对照的缺失Chlb的突变体大麦Chlorina-f2[2]都于3月初播种于实验田中。　　每个样品取30g新鲜的叶片,先用自来水后用蒸馏水冲洗干净。把洗净的叶片摊放在干净的纱布上吸干表面水分,剪碎,加入100mL预冷的含有0.4mol/L山梨醇、0.1mol/LTris-HCl(pH7.6)的缓冲液,用组织捣碎机先慢速捣碎1…     

Study of polynucleotide conformation by resolution-enhanced ultraviolet spectroscopy poly(rC) and poly(dC).

Self-deconvolution and the fourth derivative of ultraviolet absorption spectra have been used to study stacked single-stranded and double-helix structures of different cytosine-containing polynucleotides for the first time. These compounds were studied under different solution conditions (pH and organic solvents) and at low temperatures. The red shift of the lower band (B2u band plus possibly some n-->pi* transition) of the absorption spectra in the cytosine-containing polynucleotides and the appearance of new peaks in the deconvoluted and derivative spectra in the 280-310 nm region are attributed mainly to cytosine-cytosine stacking interactions. In particular, the fourth-derivative peaks at wavelengths higher than 290 nm can be associated to coupling of electronic transitions of cytosine bases. The nature of the electronic transitions producing the absorption bands which are resolved in the aforementioned fourth-derivative peaks is discussed. It is concluded that the resolution-enhancement techniques used in this work, i.e. self-deconvolution and fourth derivative, complement each other and are useful methods to study structural changes of single-stranded and double-stranded polynucleotides allowing, at the same time, more information to be obtained about specific stacking interactions than classical absorption spectrophotometry.     

2-十三烷酮对棉铃虫细胞色素P450的诱导作用

将2-十三烷酮按0.005%～0.01%（重量比）的浓度加到棉铃虫人工饲料中,连续诱导3代,测定棉铃虫中肠和脂肪体中细胞色素P450(cyt-P450)含量以及与标准配基（正丁醇、吡啶、苯胺、环己烷）形成的氧化型结合光谱。2-十三烷酮诱导品系的中肠cyt-P450与CO结合光谱的最大吸收峰在449 nm处,脂肪体cyt-P450与CO结合光谱的最大吸收峰在450.7 nm处。中肠cyt-P450除了在450 nm附近存在一个吸收峰外,在通入CO后依次在414、415、418 nm附近出现吸收峰,随后该峰消失,随着时间的推移（第31次扫描）在420 nm处又开始出现一个弱吸收峰。2-十三烷酮诱导品系的中肠、脂肪体cyt-P450与4种标准配基形成的差光谱与对照相比在峰型上存在着不同程度的差异。中肠cyt-P450与正丁醇形成双峰双谷的光谱;脂肪体cyt-P450与正丁醇形成的光谱最大吸收峰在416.61 nm处,波谷在424.91 nm处;中肠cyt-P450和脂肪体cyt-P450与吡啶形成的光谱为典型的Ⅱ型光谱,而与环己烷形成的光谱为不典型Ⅰ型光谱;中肠和脂肪体的cyt-P450与苯胺形成典型的Ⅱ型光谱,最大吸收峰分别在443.30和428.92 nm处,最小吸收分别在402.30和401.00 nm处。     

Substrate-induced spectral changes in human normal and chronic myeloid leukemic granulocytes

Several drugs/chemicals were allowed to interact with the cytochrome P-450 dependent mixed function oxidase system in the postmitochrondrial supernatant fractions of Ficoll-Hypaque-separated granulocytes from human normal subjects and patients with chronic myeloid leukemia. The substrate-induced spectral changes were followed by recording the difference spectra. Compounds conventionally classified as type I and type II substrates, on addition to S1 fractions of both normal and leukemic granulocytes, caused spectral changes that were reverse to those reported for the rat liver microsomes. Aminopyrine, phenobarbital, and Tween 80 evoked a reverse type I spectral change with a peak at 420-430 nm and a trough at 380-400 nm, whereas aniline and pyridine induced a modified type I (a reverse type II) spectral change characterized by a peak at 408 nm and a trough at 421 nm. These changes were found to be quantitatively proportional to the amounts of substrate added. However, the magnitude of the peaks and troughs was considerably less in the S1 fraction of the leukemic granulocytes. Correspondingly, total heme content was significantly decreased in S1 fractions of CML granulocytes as compared to similar fractions of normal granulocytes.     

Second derivative fluorescence spectra of indole compounds

The fluorescence emission spectrum of N-acetyl tryptophan amide (NATA) in 20 mM K-phosphate buffer, pH 7.5, with excitation at 295 nm, when subjected to second derivatization, showed two troughs at 340 1.0 nm (A) and 358.5 1.0 nm (B). Linear dependence of derivative intensities at A and B was observed with increasing NATA concentration between 0-30 nM but the intensity ratio (B/A), termed R, was found to be invariant at 0.70 0.05. R remained unaffected with variation of the pH (4-10), temperature (15-70 degrees C), salt concentration (0-2 M NaCl), and excitation wavelength between 280-300 nm. A 50-fold molar excess of N-acetyl tyrosine over 10 nM NATA and inclusion of a quencher like 0.8 M acrylamide, 0.4 M potassium iodide or trichloroethanol had no effect on R. It was, however, linearly dependent on the polarity of the solvent-in 1,4-dioxane it became 0.07 0.05. Derivative spectra of tryptophans of proteins largely resembled that of NATA. Low R values of between 0.02-0.34 were observed for proteins under native conditions, which is consistent with the general buried character of tryptophan residues. R increased to 0.6-0.9 after unfolding with denaturants or extensive proteolysis and decreased to close to the original value after refolding. The equilibrium unfolding transitions of proteins expressed as R largely resembled the transitions measured using other physical parameters. R appears to be a more sensitive index for monitoring the hydrophobic environment of tryptophans in protein compared to parameters like emission maxima or intensity of underivatised spectra.     

Conformational isomerizations of the poly(dA-dT) and poly(amino2dA-dT) duplexes involving the unusual X-DNA double helix: a fourth derivative spectrophotometric study

Fourth derivative spectrophotometry has been applied to monitor conformational isomerizations of polynucleotides for the first time. The transitions studied have been the B-A and A-X isomerizations of poly(dA-dT) and the B-X one of poly(amino2dA-dT). Parameters obtained from the fourth derivative spectra have been used to follow these conformational changes. The A form of poly(dA-dT) has been characterized by a new fourth derivative peak at 293.0 nm which can be associated to interstrand adenine-adenine interactions. Furthermore, some of the fourth derivative peaks in the long wavelength region (270-310 nm) can be related to stacking interactions present in the polynucleotide double helices. The tentative assignment of these peaks, particularly that at 299.0 nm in the derivative spectra of poly(amino2dA-dT), to n----pi electronic transitions is discussed.     

Effects of some divalent metal ions on the aging phenomenon of hematoporphyrin and photofrin II

Dilute aqueous solutions of hematoporphyrin (Hp) and its derivative (Hpd or PF II) have been found to undergo a transformation (aging) on keeping at room temperature leading to (i) shift of the Soret band from 395 nm to 405 nm, (ii) disappearance of visible bands I (610 nm) and IV (503 nm) and (iii) shift of the first emission band from 615 nm to 580 nm. The transformation was concentration dependent. The effects of concentration and temperature on the absorption spectra were much more pronounced in Hp than in Photofrin II (PF II). Variation of pH resulted in changes in the relative intensities of the absorption bands, possibly due to formation of different ionic species at different pH. The rate of transformation was accelerated in the presence of Zn ions (0.01 microM) and considerably increased at higher (50 microM) concentration. The effect of Cu ions was different from the effect of aging. It formed the metal-chelate even when present in very small amounts. The results (absorption and fluorescence analysis) suggest that in dilute solutions (conc. less than or equal to 2 microM) of Hp and PF II, Zn ions present in glass and water as impurity, deform the porphyrin nucleus leading to changes in the conjugated ring symmetry and hence changes in the absorption and fluorescence spectra, while in higher concentrations (greater than 2 microM) it forms the metal chelate as evidenced by their absorption and fluorescence spectra.     

Derivative absorption spectroscopy from 5—300 K of bacteriochlorophyll a-protein from Prosthecochloris aestuarii

Absorption spectra of the bacteriochlorophyll $\text{a-}\text{protein}$ from Prosthecochloris aestuarii were measured at temperatures from 2.9 to 300 K. Fourth and eight derivatives of the spectra were calculated from the digital data. From an analysis of 34 scans taken from 750 to 850 nm at 5 K, and 130 scans taken from 822 to 838 nm, we find evidence for nine peaks, six of which are probably 0-0 excitonic and three probably higher vibronic features. The major peaks are resolved in the derivative spectra to 300 K, and all shift with temperature by less than 1 nm compared to their 5 K positions, except for the 825 nm peak which shifts about 2 nm. The most prominent fourth derivative peak at 300 K shifts from 812.9 nm in the standard buffer solution to 814.1 nm in the cryogenic solution in which our low temperature measurements were made. We conclude that the conformation of the protein at 5 K is essentially the same as at 300 K.     

Visible absorption spectra of quantum mixed-spin ferric heme proteins.

Recently, it has been shown that the magnetic data for Chromatium ferricytochrome c' at pH 7 are consistent with quantum mechanically (as distinguished from thermally) mixed mid-spin (S = 3/2) and high-spin (S = 5/2) heme. Visible absorption spectra of the protein measured at 77 degrees K and 293 degrees K, pH 7, show peaks at 400, 490, and 632 nm. The observation of a 630 nm band in quantum mixed-spin heme spectra, and the spin state-dependence of the band intensity, are discussed in the context of the iron-ligand structure for quantum mixed-spin heme inferred from magnetic data.     

淀粉液化芽孢杆菌α-淀粉酶在盐酸胍和碳酸胍变性过程中的构象变化

 利用紫外差光谱,荧光光谱和圆二色谱法对比地研究了淀粉液化茅孢杆菌α-淀粉酶在盐酸胍和碳酸胍变性过程的构象变化与活性关系以及在变性早期钙离子对酶构象的稳定作用。     

Interaction of plastocyanin with oligopeptides: effect of lysine distribution within the peptide

We synthesized and purified four oligopeptides containing four lysines (KKKK, GKKGGKK, KKGGGKK, and KGKGKGK) as models for the plastocyanin (PC) interacting site of cytochrome f. These peptides competitively inhibited electron transfer between cytochrome c and PC. The inhibitory effect increased as the peptide concentrations were increased. The association constants between PC and the peptides did not differ significantly (3500-5100 M(-1)), although the association constant of PC-KGKGKGK was a little larger than the constants between PC and other peptides. Changes in the absorption spectrum of PC were observed when the peptides were added to the PC solution: peaks and troughs were detected at about 460 and 630 nm and at about 560 and 700 nm, respectively, in the difference absorption spectra between the spectra with and without peptides. These changes were attributed to the structural change at the copper site of PC by interaction with the peptides. The structural change was most significant when tetralysine was used. These results show that binding of the oligopeptide to PC is slightly more efficient when lysines are distributed uniformly within the peptide, whereas the structural change of PC becomes larger when the lysines are close to each other within the peptide.     

Spectral properties of porcine plasminogen: study of the acidic transition (author's transl)]

The acidic transition of porcine plasminogen, prepared by affinity chromatography, was studied by non-destructive methods. These methods are based on the analysis of the behaviour of the tryptophyls under various conditions. The perturbation of the absorption and emission spectra by pH or temperature and the dynamic quenching of the intrinsic fluorescence are used to obtain information on structural changes which affect the environment of these residues. It is shown that by decreasing pH the fluorescence emission spectra are shifted toward the long wavelengths, with a broadening of the fluorescence band. The same effect can be obtained at constant pH by heating the protein solution. In order to analyze these phenomena, it is assumed that the fluorescence intensities at 355 nm and 328 nm reflect the proportion of the tryptophans which are exposed to the solvent, and buried, respectively. The plot of the ratio of the fluorescence intensities at these wavelengths versus pH or temperature leads to a titration curve showing an unmasking of tryptophans. The proportion of exposed tryptophans is measured by the dynamic fluorescence quenching technique and the data analyzed according to Lehrer. The plot of the fraction of exposed tryptophyls versus pH also shows the unmasking of these chromophores. Thermal perturbation of a solution of plaminogen at neutral pH induces a difference absorption spectrum whose amplitudes at the maxima are proportional to the number of exposed aromatic residues. The comparison with a solution of fully denatured plasminogen in 6 M guanidium chloride, where all the tryptophyls are exposed, shows that the percentage of exposure is equal to 59%. This number is significantly higher than the percentage found by the fluorescence quenching technique (20%), indicating that some tryptophyls are located in crevices, exposed to the solvent but not to the iodide. At acidic pH the absorption difference spectra induced by thermal perturbation are not classical, since they show an inversion and a new band between 300 nm and 305 nm. This band is mentioned in the literature as a minor band of tryptophan which appears when this chromophore is located in an asymmetric environment. On plotting the maximum amplitude of these spectra obtained at acidic pH versus temperature, we obtain a curve indicating that two types of antagonistic interactions are involved in the perturbation of the chromophores spectra. The spectrophotometric titration of plasminogen gives classical absorption difference spectra. By plotting the maximum amplitude at 292 nm versus pH, we obtain a titration curve with an apparent pK of 2.9 units. This pK is acidic which respect to the pK value of a normal carboxyl. This low value can be due to a positively charged group in the neighbourhood of a carboxyl, which interacts with one or more chromophores. When the carboxyl becomes protonated, this positively charged group is free and available to perturb the environment of some chromophores...     

The ultraviolet absorption spectra of chromatoid bodies of Entamoeba invadens in situ : An optical titration

Ultraviolet absorption spectra of chromatoid bodies, which have been considered to be crystals of ribosomes, found in the cysts of E. invadens, were obtained by microspectrophotometry. The absorption spectra changed from a single 275 nm peak obtained from fresh cysts to peaks at 260 nm and 310 nm obtained from one day old cysts. Three day old cysts showed a further change to two peaks at 260 nm and 360 nm. We have attributed these changes to the oxidation of tryptophan to formylkynurenine and the hydrolysis of formylkynurenine to kynurenine.     

Conformational Isomerizations of the Poly(dA-dT) and Poly(amino2dA-dT) Duplexes Involving the Unusual X-DNA Double Helix: A Fourth Derivative Spectrophotometric Study

Abstract

Fourth derivative spectrophotometry has been applied to monitor conformational isomerizations of polynucleotides for the first time. The transitions studied have been the B-A and A-X isomerizations of poly(dA-dT) and the B-X one of poly(amino²dA-dT). Parameters obtained from the fourth derivative spectra have been used to follow these conformational changes. The A form of poly(dA-dT) has been characterized by a new fourth derivative peak at 293.0 nm which can be associated to interstrand adenine-adenine interactions. Furthermore, some of the fourth derivative peaks in the long wavelength region (270–310 nm) can be related to stacking interactions present in the polynucleotide double helices. The tentative assignment of these peaks, particularly that at 299.0 nm in the derivative spectra of poly(amino²dA-dT), to n→π^* electronic transitions is discussed.