Spectrophotometric Analysis of Pigments: A Critical Assessment of a High-Throughput Method for Analysis of Algal Pigment Mixtures by Spectral Deconvolution

The Gauss-peak spectra (GPS) method represents individual pigment spectra as weighted sums of Gaussian functions, and uses these to model absorbance spectra of phytoplankton pigment mixtures. We here present several improvements for this type of methodology, including adaptation to plate reader technology and efficient model fitting by open source software. We use a one-step modeling of both pigment absorption and background attenuation with non-negative least squares, following a one-time instrument-specific calibration. The fitted background is shown to be higher than a solvent blank, with features reflecting contributions from both scatter and non-pigment absorption. We assessed pigment aliasing due to absorption spectra similarity by Monte Carlo simulation, and used this information to select a robust set of identifiable pigments that are also expected to be common in natural samples. To test the method’s performance, we analyzed absorbance spectra of pigment extracts from sediment cores, 75 natural lake samples, and four phytoplankton cultures, and compared the estimated pigment concentrations with concentrations obtained using high performance liquid chromatography (HPLC). The deviance between observed and fitted spectra was generally very low, indicating that measured spectra could successfully be reconstructed as weighted sums of pigment and background components. Concentrations of total chlorophylls and total carotenoids could accurately be estimated for both sediment and lake samples, but individual pigment concentrations (especially carotenoids) proved difficult to resolve due to similarity between their absorbance spectra. In general, our modified-GPS method provides an improvement of the GPS method that is a fast, inexpensive, and high-throughput alternative for screening of pigment composition in samples of phytoplankton material.     

An improved method for estimating R-phycoerythrin and R-phycocyanin contents from crude aqueous extracts of <Emphasis Type="Italic">Porphyra</Emphasis> (Bangiales,Rhodophyta)

One frequently-cited method for determining phycoerythrin (PE) and phycocyanin (PC) contents from crude aqueous extracts of red seaweeds utilizes peaks and troughs of absorbance spectra. The trough absorbance values are used to establish a linear or logarithmic baseline attributable to background scatter of particulate cellular debris not removed by centrifugation. Pigment contents are calculated by subtracting baseline values from PE and PC absorbance peaks. The baseline correction is intended to make the method independent of centrifugation time and/or speed. However, when crude extracts of Porphyra were analyzed using this protocol, R-PE and R-PC estimates were significantly affected by centrifugation time, suggesting that the method was not reliable for the genus. The present study has shown that with sufficient centrifugation, background scatter in Porphyra extracts can be removed, the remaining spectrum representing the overlapping absorbance peaks of water-soluble pigments in the extract. Using fourth derivative analysis of Porphyra extract absorbance spectra, peaks corresponding to chlorophyll, R-PE, R-PC, and allophycocyanin (APC) were identified. Dilute solutions of purified R-PE, R-PC and chlorophyll were scanned separately to identify spectral overlaps and develop new equations for phycobilin quantification. The new equations were used to estimate R-PE and R-PC contents of Porphyra extracts and purified R-PE, R-PC and chlorophyll solutions were mixed according to concentrations corresponding to the sample estimates. Absorbances and fourth derivative spectra of the sample extract and purified pigment mixtures were compared and found to coincide. The newly derived equations are more accurate for determining R-PE and R-PC of Porphyra than previously published methods.     

A routine method to determine the chlorophyll a,pheophytin a and β-carotene contents of isolated Photosystem II reaction center complexes

The most simple way in which the stoichiometry of chlorophyll a, pheophytin a and -carotene in isolated Photosystem II reaction center complexes can be determined is by analysis of the spectrum of the extracted pigments in 80% acetone. We present two different calculation methods using the extinction coefficients of the purified pigments in 80% acetone at different wavelengths. One of these methods also accounts for the possible presence of chlorophyll b. The results are compared with results obtained with HPLC pigment analysis, and indicate that these methods are suitable for routine determination of the pigment stoichiometry of isolated Photosystem II reaction center complexes.     

The formation of Zn-Chl a in Chlorella heterotrophically grown in the dark with an excessive amount of Zn2+

Chlorella, when heterotrophically cultivated in the dark, is able to grow with Zn2+ at 10-40 mM, which is 10 times the concentration lethal to autotrophically grown cells. However, the lag phase is prolonged with increasing concentrations of Zn2+; for example, in this study, 1 d of the control lag phase was prolonged to about 16 d with Zn2+ at 16.7 mM (x2,000 of the control). Once the cells started to grow, the log phase was finished within 4-6 d regardless of Zn concentration, which was almost the same as that of the control. The photosysystem I reaction center chlorophyll, P700, and the far-red fluorescence were detected only after the late log phase of the growth curve, suggesting that chlorophyll-protein complexes can be organized after cell division has ceased. Interestingly, at more than 16.7 mM of Zn2+, Zn-chlorophyll a was accumulated and finally accounted for about 25% of the total chlorophyll a in the late stationary phase. We found that the Zn-chlorophyll a was present in the thylakoid membranes and not in the soluble fractions of the cells. The rather low fluorescence yield at around 680 nm in the stationary phase suggests that Zn-chlorophyll a can transfer its excitation energy to other chlorophylls. Before accumulation of Zn-chlorophyll a, a marked amount of pheophytin a was temporally accumulated, suggesting that Zn-chlorophyll a could be chemically synthesized via pheophytin a.     

The effect of anions on spectral properties of iodopsin and native cones in the frog retina (microspectrophotometric study)

Absorption spectra of single outer segments of the frog Rana temporaria photoreceptors were registered. Effects of nitrate and chloride ions on spectral properties of cone and rod pigments were compared. These pigments proved to differ in structure of the native photoreceptor membrane and, therefore, in effect of hydrophile environment on the chromophore centrum. Substitution of chloride by nitrate ions led to the hypochromic shift of the cone absorption spectrum (20-25 nm) but does not affect the spectrum on case of rod pigment. The ionochromic behaviour of cone pigments resembles that of the light-sensitive halobacterium protein halorhodopsin, in native membrane. We suppose that the effect of anions on the chromophore centrum may be the cause of bathochromic shifts of absorption spectra of longwave-length retinal-containing pigments.     

Divergent mechanisms for the tuning of shortwave sensitive visual pigments in vertebrates.

Of the four classes of vertebrate cone visual pigments, the shortwave-sensitive SWS1 class shows the shortest lambda(max) values with peaks in different species in either the violet (390-435 nm) or ultraviolet (around 365 nm) regions of the spectrum. Phylogenetic evidence indicates that the ancestral pigment was probably UV-sensitive (UVS) and that the shifts between violet and UV have occurred many times during evolution. This is supported by the different mechanisms for these shifts in different species. All visual pigments possess a chromophore linked via a Schiff base to a Lys residue in opsin protein. In violet-sensitive (VS) pigments, the Schiff base is protonated whereas in UVS pigments, it is almost certainly unprotonated. The generation of VS from ancestral UVS pigments most likely involved amino acid substitutions in the opsin protein that serve to stabilise protonation. The key residues in the opsin protein for this are at sites 86 and 90 that are adjacent to the Schiff base and the counterion at Glu113. In this review, the different molecular mechanisms for the UV or violet shifts are presented and discussed in the context of the structural model of bovine rhodopsin.     

Photochromic pigments in akinetes and pigment characteristics of akinetes in comparison with vegetative cells of Anabaena variabilis

Since akinete germination is triggered by light and the action spectrum for this process has features in common with the spectra of the two photochromic pigments, phycochromes b and d, a search was made for the presence of these phycochromes in akinetes of the blue-green alga. Anabaena variabilis Kützing. Allophycocyanin-B was also looked for, since the action spectrum for akinete germination points to a possible participation of this pigment too. Isoelectric focusing was used for purification of the pigments. The different fractions were investigated for phycochromes b and d by measuring the absorbance difference spectra: for phycochrome b. 500 nm irradiated minus 570 nm irradiated, and for phycochrome d, 650 nm irradiated minus 610 nm irradiated. For determination of allophycocyanin-B. fourth derivative analysis of absorption spectra was made for some of the fractions from the isoelectric focusing column. Phycochrome b was also assayed for by measuring in vivo absorption difference spectra. The assays were positive for all three pigments. The complete photosynthetic pigment systems were also studied by in vivo fluorescence measurements on both akinetes and vegetative cells of Anabaena variabilis. Fluorescence emission and excitation spectra at selected emission wavelengths were measured at room temperature and liquid nitrogen temperature. The energy transfer from phycoerythrocyanin to phycocyanin is very efficient under all conditions, as is the energy transfer from phycocyanin to allophycocyanin at room temperature. At low temperature, however, phycocyanin is partly decoupled from allophycocyanin, particularly in the akinetes; the energy transfer from allophycocyanin to chlorophyll a is less efficient at low temperature in both types of cells, but especially in akinetes. Delayed light emission was measured for both types of cells and found to be very weak in akinetes compared to vegetative cells. From this study it would seem that akinetes lack an active photosystem II, although the 691 nm peak in the 570 nm excited low temperature fluorescence emission spectrum proves the presence of photosystem II chlorophyll, and also its energetic connection to the phycobilisomes.     

真眼点藻类色素的提取与测定方法

分别采用甲醇、乙醇和丙酮3种有机溶剂提取7种真眼点藻的色素,比较3种有机溶剂提取色素的效果,测定3种有机溶剂色素提取液的吸收光谱,利用分光光度法计算藻的叶绿素a和类胡萝卜素的含量,并比较甲醇和乙醇色素提取液在A₄₇₀和A₆₆₆的最大吸收峰。结果表明:使用乙醇比甲醇和90%丙酮操作更简便、快捷并且毒害低。3种有机溶剂色素提取液的叶绿素a和类胡萝卜素的含量均无显著性差异(P>0.05),提取率基本一致。色素在3种有机溶剂中的吸收光谱相似,甲醇和乙醇的色素提取液在A₄₇₀和A₆₆₆的最大吸收峰并无显著性差异(P>0.05)。乙醇色素提取液可使用Lichtenthaler的公式计算色素含量。     

The effect of eye colour pigments on the action spectrum of Drosophila

In vivo absorption spectra for Drosophila melanogaster eye colour pigment classes (drosopterins and ommatins) were constructed by subtracting the whole eye electroretinographic (ERG) spectral sensitivities of cn and bw respectively from the sensitivities of white-eyed strains. In situ microspectrophotometric (MSP) absorption spectra were also obtained. Both the ERG and MSP drosopterin spectra show a visible peak at 500 nm compared to the 480 nm peak of in vitro drosopterins. For the ommatins, the ERG absorption spectrum peaks at 450 nm while the MSP spectrum peaks at 400 and 525 nm. The ERG spectrum is similar to the in vitro absorption spectrum of xanthommatin while the MSP spectrum is similar to the in vitro absorption spectrum of reduced xanthommatin. The ERG absorption spectra for the drosopterins and the ommatins yield an accurate prediction of the effect of the combined pigments in wild-type eyes. Newly emerged and 7 day post-emergence bw flies show quantitatively similar pigment absorption effects while the drosopterins depress the sensitivity of newly emerged cn flies to a greater extent than that of cn flies 7 days after emergence.     

Changes in chlorophyll <Emphasis Type="Italic">a</Emphasis> and β-carotene fluorescence caused by hydrogen peroxide

Hydrogen peroxide is shown to change the fluorescence of β-carotene and chlorophyll a as well as various mixtures of the pigments in Langmuir films and in complex with bovine serum albumin in water solution. Similar effects were shown for zinc etioporphyrin II in the films. No shifts of the fluorescence peaks were registered in any case.     

Automated screening for metabolites in complex mixtures using 2D COSY NMR spectroscopy

One of the greatest challenges in metabolomics is the rapid and unambiguous identification and quantification of metabolites in a biological sample. Although one-dimensional (1D) proton nuclear magnetic resonance (NMR) spectra can be acquired rapidly, they are complicated by severe peak overlap that can significantly hinder the automated identification and quantification of metabolites. Furthermore, it is currently not reasonable to assume that NMR spectra of pure metabolites are available a priori for every metabolite in a biological sample. In this paper we develop and report on tests of methods that assist in the automatic identification of metabolites using proton two-dimensional (2D) correlation spectroscopy (COSY) NMR. Given a database of 2D COSY spectra for the metabolites of interest, our methods provide a list sorted by a heuristic likelihood of the metabolites present in a sample that has been analyzed using 2D COSY NMR. Our models attempt to correct the displacement of the peaks that can occur from one sample to the next, due to pH, temperature and matrix effects, using a statistical and chemical model. The correction of one peak can result in an implied correction of others due to spin–spin coupling. Furthermore, these displacements are not independent: they depend on the relative position of functional groups in the molecule. We report experimental results using defined mixtures of amino acids as well as real complex biological samples that demonstrate that our methods can be very effective at automatically and rapidly identifying metabolites.     

The Existence of Chlorophyll c in the Chl b-Containing,Light-Harvesting Complex of the Green Alga Mantoniella squamata (Prasinophyceae)

The prasinophycean alga Mantoniella contains, in addition to Chl a and b, at least a third green pigment which is functionally active in the light-harvesting antenna. This third Chl was isolated in order to elucidate its chemical structure. The absorption and fluorescence spectra were measured not only from the purified pigment but also from its pheophytin and its methylpheophorbide. The spectra were compared with those of authentic Chl c-1 and c-2, which were isolated from the diatom Nitzschia sp. and with Mg-DVPP (purified from Rhodobacter). The results show that the pigment from Mantoniella compares best with Chl c-1. In order to clarify the spectral data, Chl c-1 and c-2, Mg-DVPP, and the pigment from Mantoniella were subjected to a chromatographic system that is able to separate these porphyrins. The chromatographic analysis clearly shows that the pigment from Mantoniella co-migrates with Chl c-1 and not with the bacterial pigment. Mantoniella is the first organism which has been demonstrated to contain Chl a, b, and authentic c.     

Difference spectrum distortion in non-homogeneous pigment associations: Abnormal phytochrome spectra in vivo

Difference spectra for phototransformation of phytochrome in vivo may vary considerably in shape, in peak position and in the ratio of the heights of red to far-red peaks. Assuming non-homogeneous distributions of pigments, spectra could be calculated for a number of models of plant cells containing both phytochrome and chlorophyll. Some models satisfactorily account for the observed spectra and provide some information about the intracellular pigment distribution. The theory may also find application in the interpretation of difference spectra of other naturally occurring pigment associations.     

Peridinin-chlorophyll-protein reconstituted with chlorophyll mixtures: preparation, bulk and single molecule spectroscopy

Reconstitution of the 16 kDa N-terminal domain of the peridinin-chlorophyll-protein, N-PCP, with mixtures of chlorophyll a (Chl a) and Chl b, resulted in 32 kDa complexes containing two pigment clusters, each bound to one N-PCP. Besides homo-chlorophyllous complexes, hetero-chlorophyllous ones were obtained that contain Chl a in one pigment cluster, and Chl b in the other. Binding of Chl b is stronger than that of the native pigment, Chl a. Energy transfer from Chl b to Chl a is efficient, but there are only weak interactions between the two pigments. Individual homo- and hetero-chlorophyllous complexes were investigated by single molecule spectroscopy using excitation into the peridinin absorption band and scanning of the Chl fluorescence, the latter show frequently well resolved emissions of the two pigments.     

Energy and electron transfer in photosystem II of a chlorophyll b-containing Synechocystis sp. PCC 6803 mutant

Using a Synechocystis sp. PCC 6803 mutant strain that lacks photosystem (PS) I and that synthesizes chlorophyll (Chl) b, a pigment that is not naturally present in the wild-type cyanobacterium, the functional consequences of incorporation of this pigment into the PS II core complex were investigated. Despite substitution of up to 75% of the Chl a in the PS II core complex by Chl b, the modified PS II centers remained essentially functional and were able to oxidize water and reduce Q(A), even upon selective excitation of Chl b at 460 nm. Time-resolved fluorescence decay measurements upon Chl excitation showed a significant reduction in the amplitude of the 60-70 ps component of fluorescence decay in open Chl b-containing PS II centers. This may indicate slower energy transfer from the PS II core antenna to the reaction center pigments or slower energy trapping. Chl b and pheophytin b were present in isolated PS II reaction centers. Pheophytin b can be reversibly photoreduced, as evidenced from the absorption bleaching at approximately 440 and 650 nm upon illumination in the presence of dithionite. Upon excitation at 685 nm, transient absorption measurements using PS II particles showed some bleaching at 650 nm together with a major decrease in absorption around 678 nm. The 650 nm bleaching that developed within approximately 10 ps after the flash and then remained virtually unchanged for up to 1 ns was attributed to formation of reduced pheophytin b and oxidized Chl b in some PS II reaction centers. Chl b-containing PS II had a lower rate of charge recombination of Q(A)(-) with the donor side and a significantly decreased yield of delayed luminescence in the presence of DCMU. Taken together, the data suggest that Chl b and pheophytin b participate in electron-transfer reactions in PS II reaction centers of Chl b-containing mutant of Synechocystis without significant impairment of PS II function.     

Molecular organization of pheophytin a in aqueous solutions of detergents

Absorption, fluorescence and excitation fluorescence spectra of pheophytin a have been measured in aqueous solutions of nonionic (Triton X-100), anionic (sodium lauryl sulphate) and cationic (Cetyl pyridinium chloride) detergents at different concentrations and pH after system relaxation. By measuring the second derivative and differential spectra, it has been shown, that if detergent concentrations are lower than critical micelles concentration, or if the detergent is completely absent, the pigment forms conglomerates containing both dimeric and monomeric forms with an efficient energy transfer between them. If detergent concentrations are higher than critical micelles concentration, pheophytin a localizes in detergent micelle in monomeric form at neutral and acidic pH, and allomerizes at alkaline pH. The spectral characteristics of pheophytin a dimers in the conglomerate and its monomers in micelles poorly (if at all) depend on the sign of the detergent molecule charge.     

The pigment system of the photoreceptor 7 yellow in the fly,a complex photoreceptor

Summary The 7y photoreceptor in the fly (Musca, Calliphora) retina harbours an unusually complex pigment system consisting of a bistable visual pigment (xanthopsin, X and metaxanthopsin, M), a blue-absorbing C₄₀-carotenoid (zeaxanthin and/or lutein) and a uv sensitizing pigment (3-OH retinol).The difference spectrum and photoequilibrium spectrum in single 7y rhabdomeres were determined microspectrophotometrically (Fig. 2).The extinction spectrum of the C₄₀-carotenoid has a pronounced vibrational structure, with peaks at 430, 450 and 480 nm (Fig. 3). The off-axis spectral sensitivity, determined electrophysiologically with 1 nm resolution shows no trace of this fine structure thus excluding the possibility that the C₄₀-carotenoid is a second sensitizing pigment (Fig. 4).The absorption spectra of X and M are derived by fitting nomogram spectra (based on fly R1–6 xanthopsin) to the difference spectrum. max for X is 425 nm, and for M 510 nm (Fig. 5). It is shown that the photoequilibrium spectrum and the difference spectrum can be used to derive the relative photosensitivity spectra of X and M using the analytical method developed by Stavenga (1975). The result (Fig. 6) shows a pronounced uv sensitivity for both, X and M, indicating that the uv sensitizing pigment transfers energy to both X and M. A value of 0.7 for, the relative efficiency of photoconversion for X and M, is obtained by fitting the analytically derived relative photosensitivity spectra to the absorption spectra at wavelengths beyond 420 nm.     

毛竹茎秆叶绿体超微结构及其发射荧光光谱特征

为了探讨毛竹（Phyllostachys pubescens）茎秆的光合特性,以1龄和3龄毛竹为材料,观察了茎秆和叶中叶绿体的超微结构,测定了光合色素含量以及发射荧光光谱。结果表明：茎秆中叶绿体发育完整,其类囊体垛叠程度高于叶,并含有淀粉粒。茎秆中叶绿素总含量、类胡萝卜素及Chla／b含量显著低于叶（FI〈O．05）。茎秆发射荧光光谱在735nm处没有明显的主峰,1龄和3龄毛竹茎秆光系统lI与光系统I的半峰宽比值分别比叶降低了7．0％和11．3％（P〈0．05）,峰高比值比叶分别增加了6．5％和18．3％（P〈0．05）。四阶导数光谱在650—800nm波长范围内出现了6个极大值,代表LHCII、CP43、CP47、RCI和ILHCI的发射荧光峰以及PSI和PSII的发射荧光副振峰：其中,茎秆中RCI和LHCI特征发射荧光峰与叶相比有不同程度的红移。表明毛竹茎秆叶绿体通过提高Chlb的相对含量和增加类囊体垛叠以及降低LHCI含量,来适应毛竹茎秆以红光为主的光环境。进而协调激发能在2个光系统间的分配。     

Isolation and Characterization of Carotenoid Pigments of Micrococcus roseus

In addition to canthaxanthin, seven pigment fractions were isolated from Micrococcus roseus. They were purified by solvent partitioning and by column and thin-layer chromatography. Visible absorption spectra, chromatographic behavior, and partition coefficients of the pigments and derivatives prepared from the pigments were used in characterizing them. Both alpha- and beta-carotene derivatives were present. The structure of one pigment was suggested as phoenicoxanthin (3-hydroxy-4,4'-diketo-beta-carotene). Four other pigments were tentatively characterized as a dihydroxy-3,4-dehydro-alpha-carotene, a dihydroxy-alpha-carotene, a diketo-alpha-carotene, and a polyhydroxy-beta-carotene. Two pigments were isolated in trace amounts and could not be characterized. All the pigments studied were isolated as mixtures of cis-trans isomers and all except the diketo-alpha-carotene were isolated as esters from M. roseus. Quantitation of the pigments showed that canthaxanthin (4,4'-diketo-beta-carotene) represented 85% of the pigment recovered from extracts. Three of the other pigments contributed a significant proportion of the remaining pigments, whereas the other four were present in only small amounts. beta-Carotene derivatives comprised 96% and alpha-carotene derivatives 4% of the pigments recovered from extracts.