An improved phycobilin extraction method

One unique feature in cyanoprokaryotes, rhodophytes, and cryptophytes is the presence of phycobilin pigments-these water soluble pigments can absorb red, orange, yellow, and green light enhancing the spectral range available for cellular conversion to chemical energy. The presence of phycobilin pigment complexes can be detected using fluorescence, or absorbance measures. Efficient detection of these compounds is essential for use in calibrating absorbance in remote sensing or in physiological studies. The standard procedure for phycobilin analysis involves sonication, extraction in buffer potentially coupled with additional digestion steps using enzymes, repeated freeze and thawing cycles, followed by filtration, and spectrophotometric analyses. An alternative method, using asolectin-CHAPS ((3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid – AC)) solution for extraction, and three cycles of freeze/thawing/sonication, was compared to the phosphate buffer (PB) standard procedure. Cultures of both coccoid and filamentous cyanoprokaryota had improved extraction efficiency (38–80%) using AC. After two complete extractions, no pigment was detectable in AC and near baseline fluorescence was observed in the cell pellet, whereas the PB extraction method removed <90% of the phycobilins after two extractions. Phycocyanin concentration measured by AC extraction was better correlated to lipophilic pigment concentration than using phosphate buffer extraction. AC buffered to pH 6.7 was more effective than AC 3.75. One potential source of experimental error was determined to be the use of a baseline correction for the extraction buffer, not the sample.     

红龙草红色素稳定性的研究

分析了pH值、温度、光、过氰化氢、亚硫酸钠、Vc、葡萄糖、蔗糖和苯甲酸钠等对红龙草（Altemanthera dentate ‘Ruliginosa’）红色素稳定性的影响。结果表明,红龙草红色素对热的耐受性较强,但耐光照和耐氧化性较差,且还原剂亚硫酸钠对其也有微弱的影响;在不同的pH值条件下,其吸收峰没有改变,最大吸收波长为530nm;Vc和蔗糖对该色素没有破坏作用,并有一定的护色效果;葡萄糖和苯甲酸钠对该色素也无明显影响。     

Action Spectra for Conversions of Phycochrome c from Nostoc muscorum

The reversibly photochromic pigment, phycochrome c, was extracted from the blue-green alga Nostoc muscorum strain A. Action spectra were determined for in vitro conversions of the pigment from the short wavelength to the long wavelength form and vice versa. The action peak for the absorbance decrease at 650 nm is at 630 nm. During this decrease there is only a slight increase of the absorbance in the green region. Green and yellow light (maximum efficiency at 580 nm) completely restores absorbance at 650 nm. The observations are explained by the existence of three spectrally different forms of phycochrome c: P^c₆₃₀ and P^c₆₅₀ which equilibrate in darkness and P^c₅₈₀ which is reversibly photoconvertible to P^c₆₃₀. We have also measured the absorbance changes brought about by saturating irradiations with light of various wavelengths (“photostationary state spectrum”). Extreme photostationary states were obtained with about 650 nm and 500 nm light.     

Observation of non-linear biomass-capacitance correlations: reasons and implications for bioprocess control

Electrical capacitance has been discussed as a real time measure for living biomass concentration in technical bioreactors such as brewery (fermentation) tanks. Commonly, a linear correlation between biomass concentration and capacitance is assumed. While following the growth and subsequent lipid formation of the yeast Arxula adeninivorans we observed non-linearity between biomass concentration and capacitance. Capacitance deviation from linearity coincided with incipient lipid formation and depended on the intracellular lipid content. As the extent of deviation between capacitance and biomass concentration was proportional to the lipid concentration, it was considered as a quantitative measure of intracellular product formation. The correlation between shifts in dielectric relaxation (summarized as characteristic frequency of the Cole-Cole equation) and lipid content could not be explained by interfacial polarization on the lipid droplets alone. However, the parameters of the Cole-Cole equation were found to be a clear indicator for different phases of growth and lipid production. Integrating all results in a redundancy analysis (RDA), we were able to accurately describe the formation of cellular lipid inclusions. Our measurements are thus potentially valuable as components of future bioprocess control strategies targeting intracellular products such as proteins or biopolyesters.     

Protein solubility modeling.

A thermodynamic framework (UNIQUAC model with temperature dependent parameters) is applied to model the salt-induced protein crystallization equilibrium, i.e., protein solubility. The framework introduces a term for the solubility product describing protein transfer between the liquid and solid phase and a term for the solution behavior describing deviation from ideal solution. Protein solubility is modeled as a function of salt concentration and temperature for a four-component system consisting of a protein, pseudo solvent (water and buffer), cation, and anion (salt). Two different systems, lysozyme with sodium chloride and concanavalin A with ammonium sulfate, are investigated. Comparison of the modeled and experimental protein solubility data results in an average root mean square deviation of 5.8%, demonstrating that the model closely follows the experimental behavior. Model calculations and model parameters are reviewed to examine the model and protein crystallization process.     

Constitutive changes in pigment concentrations: implications for estimating isoprene emissions using the photochemical reflectance index

The photochemical reflectance index (PRI), through its relationship with light use efficiency (LUE) and xanthophyll cycle activity, has recently been shown to hold potential for tracking isoprene emissions from vegetation. However, both PRI and isoprene emissions can also be influenced by changes in carotenoid pigment concentrations. Xanthophyll cycle activity and changes in carotenoid concentrations operate over different timescales, but the importance of constitutive changes in pigment concentrations for accurately estimating isoprene emissions using PRI is unknown. To clarify the physiological mechanisms behind the PRI–isoprene relationship, the light environment of potted Salix viminalis (osier willow) trees was modified to induce acclimation in photosynthetic rates, phytopigments, isoprene emissions and PRI. Acclimation resulted in differences in pigment concentrations, isoprene emissions and PRI. Constitutive changes in carotenoid concentration were significantly correlated with both isoprene emissions and PRI, suggesting that the relationship between PRI and isoprene emissions is significantly influenced by constitutive pigment changes. Consequently knowledge regarding how isoprene emissions are affected by both longer term changes in total carotenoid concentrations and shorter term dynamic adjustments of LUE is required to facilitate interpretation of PRI for monitoring isoprene emissions.     

Purification and physiochemical characterization of melanin pigment from Klebsiella sp. GSK

The bacterium capable of producing melanin pigment in the presence of L-tyrosine was isolated from crop field soil sample and identified as Klebsiella sp. GSK based on morphological, biochemical and 16S rDNA sequencing. The polymerization of this pigment occurs outside the cell wall, which has granular structure as melanin ghosts. The chemical characterization of pigment particles showed acid resistant, alkali soluble, insoluble in most of the organic solvents and water. The pigment gets bleached when subjected to the action of oxidants as well as reductants. This pigment was precipitated with FeCl3, ammoniacal silver nitrate and potassium ferricynide. The pigment showed high absorbance in the UV region and decreased absorbance when shifted towards the visible region. The melanin pigment was further charecterized by FT-IR and EPR spectroscopy. A key enzyme 4-hydroxyphenylacetic acid hydroxylase catalyzes the formation of melanin pigment by hydroxylation of L-tyrosine was detected in this bacterium. Inhibition studies with specific inhibitor kojic acid and KCN proved that melanin is synthesized by DOPA-Melanin pathway.     

水溶性红曲黄色素的表征及其稳定性

以含硫化合物还原红曲色素,红斑素和红曲红素分子中的环羰基还原成羟基,得到水溶性红白曲黄色素.以 UV-Vis、IR和(?)H-~(13)CNMR对其结构进行了表征.该色素的吸光度随pH增大而略呈上升趋势,避光可明显提高其稳定性.除Mg~(2 )在pH9下使其溶液产生沉淀外,Sn~(2 )、Ca~(2 )、Fe~(3 )和AI~(3 )均不影响其稳定性.不同pH和温度下加热实验结果表明,酸性条件对其热稳定性影响较大.     

Simultaneous measurement of optical rotation dispersion and absorption spectra for chiral substances

We present a new method based on optical null methods for simultaneously measuring the optical rotatory dispersion (ORD) and absorption spectra of chiral substances. The optical rotation angle at a specific wavelength can be obtained from the optical nulls of the Malus curves with and without the sample. We use the optical nulls of the two curves as benchmark points and the readings to the right of the benchmark points by a certain angular offset to eliminate the influence of the analyzer on the light intensity and obtain the absorbance of the chiral substance at a specific wavelength. The 4096 pixels of the line scan CCD can measure multiple wavelengths simultaneously so that continuous ORD and absorption spectra can be obtained. The experimental results show that the standard deviation of the specific optical rotation is 0.11 deg mL g⁻¹ dm⁻¹, the standard deviation of the maximum absorption wavelength is 0.45 nm, and that absorbance of the sample varies linearly with the concentration. This method is helpful for simplifying the experiment and has a profound influence on the analysis of the contents and molecular configurations of chiral substances in the future.     

Light scattering in TIRF microscopy: A theoretical study of the limits to surface selectivity

In TIRF microscopy, the sample resides near a surface in an evanescent optical field that, ideally, decreases in intensity with distance from the surface in a pure exponential fashion. In practice, multiple surfaces and imperfections in the optical system and refractive index (RI) inhomogeneities in the sample (often living cells) produce propagating scattered light that degrades the exponential purity. RI inhomogeneities cannot easily be avoided. How severe is the consequent optical degradation? Starting from Maxwell’s equations, we derive a first-order perturbative approximation of the electric field strength of light scattered by sample RI inhomogeneities of several types under coherent evanescent field illumination. The approximation provides an expression for the scattering field of any arbitrary RI inhomogeneity pattern. The scattering is not all propagating; some is evanescent and remains near the scattering centers. The results presented here are only a first-order approximation, and they ignore multiple scattering and reflections off the total internal reflection (TIR) surface. For simplicity, we assume that the RI variations in the z direction are insignificant within the depth of the evanescent field and consider only scattering of excitation light, not fluorescence emission light. The general conclusion of most significance from this study is that TIR scattering from a sample with RI variations typical of those on a cell culture alters the effective thickness of the illumination to only ∼50% greater than it would be without scattering. The qualitative surface selectivity of TIR fluorescence is largely retained even in the presence of scattering. Quantitatively, however, scattering will cause a deviation from the incident exponential decay at shorter distances, adding a slower decaying background. Calculations that assume a pure exponential decay will be approximations, and scattering should be taken into account. TIR scattering is only slightly dependent on polarization but is strongly reduced for the highest accessible incidence angles.     

Kaempferol 3-O-Galactoside, 7-O-Rhamnoside is the Major Green Fluorescing Compound in the Epidermis of Vicia faba

The vacuoles of lower epidermal strips from Vicia faba exhibit an intrinsic green fluorescence when incubated in alkaline buffers. Using an alkaline-induced absorbance change as a spectrophotometric assay, the major pigment responsible for this fluorescence was isolated and identified as the flavonoid: kaempferol 3-O-galactoside, 7-O-rhamnoside. The aqueous absorption maxima were 394 and 341 nanometers at pH 10.0 and 6.0, respectively, with a pKa of 8.3 and the fluorescence emission maximum was 494 nanometers at pH 10.0. The in vivo concentration was estimated to be between 3 and 10 micromolar. The absorption spectrum of this flavonoid is different from the action spectrum for stomatal opening indicating that this compound is not the photoreceptor pigment for the blue light response of Vicia faba guard cells.     

Detection of the photoactive protochlorophyllide-protein complex in the light during the greening of barley.

A photoactive protochlorophyllide-protein complex with absorbance and fluorescence maxima at 648 and 653 nm was detected in greening barley leaves without any re-darkening. The variations of the amplitudes of the absorbance and the fluorescence of the photoactive protochlorophyllide with greening time at two different light intensities indicate a close relationship between the rate of chlorophyll synthesis and the amount of the complex during the first hours. The chlorophyllide resulting from photoreduction during greening has an absorbance maximum at 684 nm, which shifts towards a shorter wavelength within a few seconds, indicating rapid liberation of the pigment from the enzyme. We conclude that chlorophyll accumulation proceeds through continuous regeneration and phototransformation of the photoactive complex.     

Mathematical modeling of elution curves for a protein mixture in ion exchange chromatography and for the optimal selection of operational conditions

Elution curves in ionic exchange chromatography (IEC) for a three-protein mixture (alpha-lactoalbumin, ovalbumin, and beta-lactoglobulin), carried out under different flow rates and ionic strength conditions, were simulated using two different mathematical models. These models were the Plate Model and the more fundamentally based Rate Model. Relatively low protein concentrations were used to avoid protein-protein interactions. Simulated elution curves were compared with experimental data not used for parameter identification. Deviation between experimental data and the simulated curves using the Plate Model was less than 0.0189 (absorbance units); a slightly higher deviation [0.0252 (absorbance units)] was obtained when the Rate Model was used. A cost function was built that included the effect of the different production stages, namely fermentation, purification, and concentration. These considered the effect on the performance of IEC; yield, purity, concentration and the time needed to accomplish the separation. Operational conditions in the IEC such as flow rate, ionic strength gradient and the operational time can be selected using this model in order to find the minimum cost for the protein production process depending on the characteristics of the final product desired such as purity and yield. This cost function was successfully used for the selection of the operational conditions as well as the fraction of the product to be collected (peak cutting) in IEC. It can be used for protein products with different characteristics and qualities, such as purity and yield, by choosing the appropriate parameters.     

High Light-Induced Unstacking of Thylakoids and Randomization of Pigment-Protein Complexes of Chloroplast

Using absorbance changes at 540 nm and the per cent chlorophyll in 10K pellet after digitonin fractionation as the criteria of thylakoid stacking, high light induced unstacking of thylakoids and randomization of pigment protein complexes of chloroplast was characterized. Cation-induced increase in absorbance at 540 nm was abolished when chloroplasts were exposed to high light. Also, the high light induced decrease in absorbance at 540 nm was more pronounced In low salt chloroplasts as compared to the decrease in 540 nm absorbance in chloroplasts isolated in high salt. The decrease In the fraction of chlorophyll in digitonin fractionated 10K pellet was much more than the decrease in chlorophyll in 10K pellet from high salt chloroplasts exposed to high light These results suggest that high light causes unstacking of thylakoids.     

Estimating cell concentration in the presence of suspended solids: a light scatter technique

A novel sensor was developed, based on light scatter, to estimate the cell concentration in the presence of suspended solids. The light scatter properties of cells in the presence of suspended solids were investigated. Two crucial observations were made: first, that the light scatter from cells is essentially a linear function of cell concentration and, second, that invariant regions are present in the light scatter spectrum of cell/solid substrate mixtures. Invariant regions are wavelength intervals of the light scatter spectrum in which the light scatter reading is independent of solid substrate concentration and only a function of cell concentration. The occurrence of invariant regions is the key behavior which allowed the quantification of cell concentration in the presence of suspended solids.An algorithm was developed for the estimation, from light scatter data, of cell concentration in the presence of solid substrate. The light scatter approach was validated by comparing cell concentrations estimated by this technique to those obtained from DNA and carbon dioxide evolution rate measurements during a series of fermentations. The model system used was Bacillus subtilis var sakainensis ATCC 21394 growing on fishmeal as the sole nitrogen source.A model was developed based on the interactions of scatter and absorbance. This model reflects the hypothesis that invariant regions are caused by changes in the absorbance of the solid substrate as a function of wavelength. (c) 1992 John Wiley & Sons, Inc.     

Light absorption by carotenoid peridinin in zooxanthellae cell and setting down of hermatypic coral to depth

Carotenoid peridinin absorbs ocean light which could penetrate deep into the water. Absolute and relative contents of symbiotic dinoflagellatae zooxanthellae are increased with depth of habitat of germatypic corals. To estimate whether the presence of peridinin in corals is chromatic adaptation or not, the absorbance of solar radiation by different amounts of peridinin and chlorophyll in natice zooxanthellae cells was evaluated. Calculations have shown that at the great depths the peredinin absorbance corresponds to 42% of total cell absorbance and that the increase of light absorbance correlating with changes of its spectral characteristics is entirely determined by presence of this carotenoid. The increase of amount of peridinin in cell is as much important as important the increase of all other pigments taken together. However, at the same time selective and preferential accumulation of peridinin and the change of its native state in the limits naturally occurred in zooxanthellae cells have only low impact on the light absorbance. The presence of peridinin could be considered as manifestation of chromatic adaptation of organism. The comparison of light absorption by zooxanthellae with different content of peridinin (or without peridinin) reveals that this pigment expands the habitat of hermatypic corals in ocean waters at 8-17 meters into the deep.     

Trichromatic visual system in an insect and its sensitivity control by blue light

Summary The spectral sensitivity of the visual cells in the compound eye of the mothDeilephila elpenor was determined by electrophysiological mass recordings during exposure to monochromatic adapting light. Three types of receptors were identified. The receptors are maximally sensitive at about 350 nm (ultraviolet), 450 nm (violet), and 525 nm (green). The spectral sensitivity of the green receptors is identical to a nomogram for a rhodopsin with _max at 525 nm. The spectral sensitivity of the other two receptors rather well agrees with nomograms for corresponding rhodopsins. The recordings indicate that the green receptors occur in larger number than the other receptors. The ultra-violet and violet receptors probably occur in about equal number.The sensitivity after monochromatic adapting illumination varies with the wavelength of the adapting light, but is not proportional to the spectral sensitivity of the receptors. The sensitivity is proportional to the concentration of visual pigment at photoequilibrium. The equilibrium is determined by the absorbance coefficients of the visual pigment and its photoproduct at each wavelength. The concentration of the visual pigment, and thereby the sensitivity, is maximal at about 450 nm, and minimal at wavelengths exceeding about 570 nm.The light from a clear sky keeps the relative concentration of visual pigment in the green receptors, and the relative sensitivity, at about 0.62. The pigment concentration in the ultra-violet receptors is about 0.8 to 0.9, and that in the violet receptors probably about 0.6. At low ambient light intensities a chemical regeneration of the visual pigments may cause an increase in sensitivity. At higher intensities the concentrations of the visual pigments remain constant. Due to the constant pigment concentrations the input signals from the receptors to the central nervous system contain unequivocal information about variations in intensity and spectral distribution of the stimulating light.The work reported in this article was supported by the Swedish Medical Research Council (grant no B 73-04X-104-02B), by Karolinska Institutet, and by a grant (to G. Höglund) from Deutscher Akademischer Austauschdienst, and by the Deutsche Forschungsgemeinschaft, Schwerpunktsprogramm Rezeptorphysiologie HA 258-10, and SFB 114.     

Scanning electron microscopy and microspectrophotometry of the photoreceptors of ictalurid catfishes

The retinal photoreceptors from larval channel catfish (Ictalurus punctatus) were studied using single cell, in situ microspectrophotometry. Rods appear at 5 days after hatch; cones are present from day one. The rods contain a visual pigment which absorbs light maximally at 540 nm. The cones contain either a green sensitive visual pigment with peak absorbance at 535 nm or a red sensitive visual pigment with peak absorbance at 608 nm. All pigments are based on vitamin A₂. Visual pigment complement does not change with age, as photoreceptors from adultI. punctatus, I. catus andI. melas contain visual pigments virtually identical to those of the larvalI. punctatus. Regardless of age, no visual pigment with peak absorbance in the short wavelength region of the spectrum was ever observed. Scanning electron microscopy of adultI. punctatus retinas showed large rods with long, cylindrical outer segments and smaller cones with short, tapered outer segments. The myoids of both rods and cones are extensable. The rods, embedded in a granular tapetal material, comprise from 50 to 60% of the photoreceptors. Only single cones are present. The data are consistent with the idea that the ictalurid catfishes spend their entire lives in an environment deficient in blue light.     

Additive-Multiplicative Approximation of Genotype-Environment Interaction

A model of genotype-environment interaction in quantitative traits is considered. The model represents an expansion of the traditional additive (first degree polynomial) approximation of genotypic and environmental effects to a second degree polynomial incorporating a multiplicative term besides the additive terms. An experimental evaluation of the model is suggested and applied to a trait in Drosophila melanogaster. The environmental variance of a genotype in the model is shown to be a function of the genotypic value: it is a convex parabola. The broad sense heritability in a population depends not only on the genotypic and environmental variances, but also on the position of the genotypic mean in the population relative to the minimum of the parabola. It is demonstrated, using the model, that GXE interaction rectional may cause a substantial non-linearity in offspring-parent regression and a reversed response to directional selection. It is also shown that directional selection may be accompanied by an increase in the heritability.     

The proteolysis adaptor,NblA, is essential for degradation of the core pigment of the cyanobacterial light‐harvesting complex

The cyanobacterial light‐harvesting complex, the phycobilisome, is degraded under nutrient limitation, allowing the cell to adjust light absorbance to its metabolic capacity. This large light‐harvesting antenna comprises a core complex of the pigment allophycocyanin, and rod‐shaped pigment assemblies emanating from the core. NblA, a low‐molecular‐weight protein, is essential for degradation of the phycobilisome. NblA mutants exhibit high absorbance of rod pigments under conditions that generally elicit phycobilisome degradation, implicating NblA in degradation of these pigments. However, the vast abundance of rod pigments and the substantial overlap between the absorbance spectra of rod and core pigments has made it difficult to directly associate NblA with proteolysis of the phycobilisome core. Furthermore, lack of allophycocyanin degradation in an NblA mutant may reflect a requirement for rod degradation preceding core degradation, and does not prove direct involvement of NblA in proteolysis of the core pigment. Therefore, in this study, we used a mutant lacking phycocyanin, the rod pigment of Synechococcus elongatusPCC7942, to examine whether NblA is required for allophycocyanin degradation. We demonstrate that NblA is essential for degradation of the core complex of the phycobilisome. Furthermore, fluorescence lifetime imaging microscopy provided in situ evidence for the interaction of NblA with allophycocyanin, and indicated that NblA interacts with allophycocyanin complexes that are associated with the photosynthetic membranes. Based on these data, as well as previous observations indicating interaction of NblA with phycobilisomes attached to the photosynthetic membranes, we suggest a model for sequential phycobilisome disassembly by NblA.