Chromatographic, spectrophotometric and mass properties of pigments separated from the chloroplast of
Abstract
Chlorophyll and carotenoid are vital components that can be found in the intrinsic part of chloroplast. Their functions include light-harvesting, energy transfer, photochemical redox reaction, as well as photoprotection. These pigments are bound non-covalently to protein to make pigment-protein supercomplex. The exact number and stoichiometry of these pigments in higher plants are varied, but their compositions include chlorophyll (Chl) a, Chl b, lutein, neoxanthin, violaxanthin, zeaxanthin and β-carotene. This chapter provides introduction to the structure and photophysical properties of these pigments, how they assemble as pigment-protein complexes and how they do their functions. Various common methods for isolation, separation and identification of chlorophylls and carotenoid are also discussed.
Keywords
- carotenoid
- chlorophyll
- core complex
- ESI-MS/MS
- high-performance liquid chromatography
- light-harvesting antenna
- photosystem
- fluorescence spectrum
1. Introduction: Why do we bother about pigments in chloroplast?
Chlorophyll and carotenoid are important pigments that have been used as intrinsic optical molecular probes to observe plant performance during different phases of development. Chlorophyll and carotenoid are biosynthesized in chloroplast and their metabolism is closely related with the chloroplast development. Chlorophyll biosynthesis begins with the formation of 5-aminolevulinic acid (ALA) from glutamate (Glu) via Glu-tRNA synthetase, Glu-tRNA reductase (GluTR) and Glu-1-semialdehyde aminotransferase (GSA-AT) [1]. Eight molecules of ALA are condensed, eventually forming the symmetric metal-free porphyrin, protoporphyrin IX (Proto IX), which is a common precursor of haem and chlorophyll. The biosynthesis of chlorophyll continues by insertion of Mg2+ into Proto IX and followed by several steps in the chlorophyll cycle to create protochlorophyllide.
Further, reaction is one of the most interesting steps because this is the first step in chlorophyll biosynthesis that requires light: the NADPH:protochlorophyllide oxidoreductase converts protochlorophyllide into chlorophyllide. This reaction is then continued to produce chlorophyll (chl)
Plant carotenoids are synthesized and accumulated exclusively in plastids, most importantly, chloroplast and chromoplast [3]. There are two types of plant carotenoid: carotene, which is cyclized and uncyclized hydrocarbons, and xanthophylls, which are oxygenated derivatives of carotenes. Carotenoid synthesis is initiated by the formation of C40 compound phytoene by the head-to-head condensation of two molecules of geranylgeranyl diphosphate (GGDP) by phytoene synthase and then to a series of 4 sequential desaturation reactions, by two separate enzymes to produce lycopene, which has 11 conjugated double bonds [4]. Lycopene is then cyclized to α-carotene or β-carotene, which is then further hydroxylated to produce colorful xanthophylls such as lutein, β-cryptoxanthin, zeaxanthin, antheraxanthin, violaxanthin and neoxanthin. The biosynthesis and accumulation of carotenoids in the dark-grown etiolated seedling are essential for the assembly of membrane structure and benefits the development of chloroplast when seedlings emerge into the light [5]. Understanding the relationship between structure and photophysical properties of these pigments can provide insights into a better study of how photosynthesis works at the molecular level in chloroplast.
2. Structure, function and photophysical properties
The photophysical properties and functions of chlorophyll and carotenoid reside in their chemical structure. Chlorophylls are defined as cyclic tetrapyrroles carrying a characteristic isocyclic five-membered ring that are functional in light-harvesting or in charge separation in photosynthesis [6]. The chemical structure with IUPAC numbering scheme of chl
Structure of carotenoid is characterized by a linear chain of conjugated π-electron double bonds (Figure 1). In oxygenic organisms, carotenoid usually contains ring structures at each end, and most carotenoids contain oxygen atoms, usually as part of hydroxyl or epoxide groups. The primary molecular factor that gives rise to their strong absorption bands in the visible spectral region is the number of π-electron conjugated double bonds, N. The position of the absorption maxima is affected by the length of the chromophore, the position of the end double bond in the chain or ring and the taking out of conjugation of one double bond in the ring or eliminating it through epoxidation. Progressive movement to longer wavelengths (bathochromic shift) is illustrated by the absorption spectra of the acyclic carotenoid of increasing chromophore length. Carotenoids show different optical characteristics in various solvents, depending on the polarizability of the solvent [9, 10]; however, generally they have a typical three-peaked absorption spectrum with well-defined maxima and minima (fine structure) (Figure 2a). A ring closure as in β-carotene produces a less-defined fine structure. The introduction of a carbonyl group in conjugation with the polyene system produces a bathochromic shift and the loss of fine structure [4]. The influence of other substituents such as OH is negligible, for example, β-carotene, cryptoxanthin and zeaxanthin all have very identical absorption spectrums. Owing to the double bonds in the molecule, all carotenoids exhibit
3. Pigment assembly in pigment protein complexes
In the chloroplast interior, there are four main constituents in plant thylakoids, that is, photosystem II (PSII), cytochrome b6f, photosystem I (PSI) and the ATP synthesis. Chlorophylls and carotenoids are embedded in PS II and PSI, large pigment-protein clusters, the structures of which are perfectly adopted to ensure that almost every absorbed photon can be utilized to drive photochemistry. Both PSII and PSI consist of two moieties, that is, core complex or the reaction center that is responsible for charge separation and light-harvesting antenna complexes that surround the core complex and have functions to increase the capture of light energy and energy transfer to the reaction center in the core complex.
One can detect chlorophyll and carotenoid bound in PSII and PSI in chloroplast by measuring their absorption and fluorescence spectra. Figure 3a (solid red line) shows the absorption spectrum of diluted chloroplast that is indicated by red shift of Chl
The current high-resolution structural models of antenna complexes have been obtained only for LHCII (2.72 Å) and recently for CP29 (2.8 Å) from PSII of spinach [17, 18]. Here we focus more on the LHCII structure. LHCII shows trimeric structure. Each monomeric contains three transmembrane α-helices, a, b and c (Figure 4a). One monomeric subunit contains eight chlorophyll (Chl)
The current high-resolution crystal structure of PS II and PSI core complexes is limited to that from cyanobacteria and from pea, respectively [21, 22]. The core of PSII is a multi-subunit complex. Most of the chromophores involve light harvesting as well as electron transfer reaction and are bound to four main subunits, that is, D1, D2, CP43 and CP47. When the core of PSII and PSI reaction center structures is compared, the arrangement of the pigments and other electron transfer co-factors is also very similar (Figure 4c and d). Here, first we look at the PSII core reaction center. The core of reaction center of PSII is made from two major polypeptides called D1 and D2; each contains five membrane-spanning α-helices. These two helices clasp each other like two cupped hands holding on to each other. The redox cofactors are arranged into two arms that lie on either side of the point where the two groups of helices interact. This arrangement of the helices and the cofactors introduces a pseudo two-fold symmetry axes that runs through the center of reaction center normal to the plane of the membrane. In Figure 4e, it is seen that the electron transport pathway in PSII begins with a pair of chlorophyll molecules called P680 (PD1 and PD2). Then each arm contains, in order, one monomeric chlorophyll molecule, one pheophytin (a chlorophyll derivative) and one plastoquinone molecule. Here, only the D1 arm is active in electron transport. Upon excitation P680 becomes oxidized and one electron is injected out and passes down the active branch to the quinone QA. P680 is re-reduced by electron transfer from a special tyrosine residue called Z (Tyrz). A second turnover of P680 delivers a second electron to the plastoquinone and the secondary quinone QB is now reduced to QBH2. The hole on Tyrz is filled by electron transfer from the manganese cluster, the oxygen evolving complex. Every four turnovers of P680 stores four positive charges in the manganese cluster that are then used to oxidize water and evolve oxygen. While in CP43 and CP47, there are a total of 49 Chl
Unlike PSII, in PS I, the same single polypeptides contain both antenna complexes (Lhca) and the reaction center core. The 3.3 Å resolution crystal structure of PSI from pea showed that plant PSI binds at least 173 Chl
The core complex of PSI is composed of smaller number of subunits (15 subunit) than PSII. The large PsaA and PsaB subunit, which contain 11 trans-membrane helices each, forms a hetero-dimer that binds ~80 Chl
4. Chromatographic isolation and identification
Chlorophyll and carotenoid can be isolated as free pigments, detached from the pigment-protein complexes, by organic solvent extraction. Important aspects such as the choice of organic solvents, light exposure and working temperature should be considered while isolating pigments. Based on the structure, chlorophyll is characterized with polar macrocycle ring with non-polar hydrocarbon tail. The structural difference between Chl
After successful isolation, liquid chromatography has been widely used as an effective technique to separate individual type of pigments and for further purification. In this technique, the pigment separation is based on the polarity which depends on the interaction of pigment with the stationary and mobile phases. Elution method either normal phase or reversed phase is chosen according to the type of pigment to be separated. In addition, the choice of liquid chromatographic methods, namely thin layer chromatography (TLC), column chromatography (CC) and high-pressure liquid chromatography (HPLC), is referred to the speed, resolution and quantity of sample [30]. Currently, ultra-fast liquid chromatography (UFLC), a recent development of HPLC, has been used as a standard for liquid chromatography to achieve high-resolution data with low time consumption [31]. Purification with non-chromatographic method has also been developed, that is, purification method using dioxane has been effective to separate chlorophyll from most of the carotenoids and some lipids [32].
Various types of column absorbents used for chromatographic separation of plant pigments have been well reviewed [30]. Here, we used a silica C30 column attached to UFLC analytic to achieve well separation of carotenoids from
Peak No | t |
λmaxs [nm] | Molecular ion | Fragment ions [ |
Identification | |||
---|---|---|---|---|---|---|---|---|
HPLC eluent | Hexane | Ethanol | Acetone | species [ |
||||
1 | 7.3 | 412,436,464 | — | — | — | — | — | Violaxanthin |
2 | 12.8 | 470,601,650 | 451,595,642 | 465,601,649 | 458,596,646 | 907.7 [M]+ | 881.7 [M – COH]+ 855.7 [M – COH – Mg]+ |
Chlorophyll |
3 | 13.4 | 422,445,472 | 422,444,473 | −,446,474 | −,448,476 | 568.4 [M]+ | 551.4 [M – OH]+ 476.4 [M – 92]+ 430.3 [M – 138]+ |
Lutein |
4 | 15.3 | −,451,477 | 425,449,478 | 425,451, 478 | 428,454,481 | 568.6 [M]+ | 476.4 [M – 92]+ | Zeaxanthin |
5 | 16.6 | 431,618,664 | 427,613,661 | 430,616,664 | 431,617,662 | 893.5 [M]+ | 871.5 [M – Mg]+ 615.2 [M – phytyl]+ |
Chlorophyll |
6 | 20.1 | 421,446,473 | 421,445,474 | 421,446,476 | 422,445,473 | 536.6 [M]+ | 445.4 [M + H – 92]+ | |
7 | 21.2 | –,452,478 | –,451,479 | –,453,480 | –,454,482 | 536.6 [M]+ | 444.5 [M – 92]+ |
Larger-scale separation of Chl
Silica and alumina are frequently used as the absorbent in the CC with the normal phase elution to separate the distinct carotenoids; however, it is not easy to use this method to separate carotenoid isomers, that is, geometrical isomers, diastereoisomers, and so on. In this case HPLC/UFLC can be used to overcome the difficulty in the separation of carotenoids by CC. Turcsi et al. (2016) revealed that the polar carotenoids including optical isomers, and region and geometrical isomers as well as non-polar carotenes, could be well separated by HPLC on C18 and C30 columns, respectively [36]. High purity of isolated pigment can be achieved by HPLC and crystallization processes. UFLC analysis of the purified zeaxanthin shows that this carotenoid had a high purity of around 99.3% (Figure 2, left). All purified pigments have purity higher than 95% (Figure 6).
Chromatographic, spectrophotometric and mass properties of pigment are minimum requirements for pigment identification [35]. These properties for all purified pigments are shown in the Table 1. In Figure 7 (right), absorption spectra of the purified chlorophyll a and the purified β-carotene in acetone have the same maximum absorption wavelength (λmax) and other spectral properties, such as the fine structure and spectrum shape, compared to these pigments in the references [37, 38]. Absorption spectrum of chlorophyll a in acetone shows typical Soret (431 nm), Qx (617 nm) and Qy (662 nm) bands, while two well-defined peaks in the absorption spectrum of β-carotene are found at 454 and 482 nm. This pigment analysis based on the results of spectrophotometer UV–Vis could support the advance pigment analysis using HPLC/UFLC equipped with photodiode array detection and coupled with the mass spectrometry. The LCMS technique has provided a power tool for pigment identification [39, 40]. Tentative identification for zeaxanthin peak separated by HPLC/UFLC analysis with PDA revealed that zeaxanthin has similar retention time (tR), maximum absorption wavelength (λmax) and the shape of absorption spectrum (data not shown) compared to the isolated zeaxanthin from corn which is a well-known source of zeaxanthin [41]. In addition the mass analysis provides the precursor and fragment ions at the specific
5. Conclusions
Chlorophyll and carotenoid are chloroplast pigments which are bound non-covalently to protein as pigment-protein complex and play a vital role in photosynthesis. Their functions include light harvesting, energy transfer, photochemical redox reaction, as well as photoprotection. The exact number and stoichiometry of these pigments in higher plants are varied, but their compositions include Chl
Acknowledgments
Tatas Hardo Panintingjati Brotosudarmo (THPB) acknowledges the competence research grant (No. 120/SP2H/LT/DRPM/IV/2017) from Kemenristekdikti for the financial support. We also acknowledge Chandra Ayu Siswanti who helped in preparation of chloroplast isolation, pigment isolation and UFLC separation works. We acknowledge Dr. Hendrik Octendy Lintang for supporting fluorescence measurements of photosystem II and I in chloroplast.
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