Metodo de pigmentos definitivo definitivo

2 downloads 0 Views 235KB Size Report
Abstract. A new HPLC method for the rapid separation of photosynthetic pigments of higher plants is reported. The method separates zeaxanthin from lutein with ...
An Improved HPLC Method for Rapid Analysis of the Xanthophyll Cycle Pigments

J. Val, E. Monge and N.R. Baker* Department of Plant Nutrition, EE Aula Dei, C.S.I.C., PO Box 202, Zaragoza, Spain. *Department of Biology, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, United Kingdom.

Abstract A new HPLC method for the rapid separation of photosynthetic pigments of higher plants is reported. The method separates zeaxanthin from lutein with a resolution power (Ri,j) of 1.77. This method uses only two isocratic procedures using a 10 cm C18 column with a flow rate of 1.5 mL min-1. Consequently this allows the use of simple HPLC systems for analyses of the pigments within a total chromatography time of 13 min. Routine and rapid analysis of the xanthophyll pigments, which have been implicated recently in the photoinhibition of photosynthesis, can be achieved with this method.

Introduction Recently considerable attention has focused on the role of carotenoids in dissipating excess excitation energy in the photosynthetic apparatus when plants are exposed to potentially photoinhibitory conditions. A close relationship between the amount of quenching of excitation energy by non-photochemical processes and the zeaxanthin content of leaves has been observed in many plants under a wide range of conditions (1). When leaves are illuminated violaxanthin (di-epoxy) is converted to zeaxanthin

(non-epoxy) via antheraxanthin

(mono-epoxy) by a de-epoxidase located in the lumen of the thylakoid, which is

thought to be activated by the light-induced low pH in the lumen (2,3). The epoxidation of zeaxanthin back to violaxanthin is catalysed by an epoxidase, which is thought to be located on the stromal side of the thylakoid membrane (4,5). With the recent increased interest in the possible role of the xanthophyll cycle in the quenching of excitation energy in leaves under stress conditions, a need has arisen for a rapid procedure for the accurate analysis of the xanthophyll pigments that does not require sophisticated gradient chromatography. The recently published HPLC methods which allow resolution of zeaxanthin from lutein, yet still produce good resolution of all the other photosynthetic pigments, require the use of sophisticated gradients, at least three solvents in mobile phase and, in some cases, long periods for the chromatography (6,7,8,9,10). In this paper we describe a new, simple HPLC method using a reversed phase C18 column which permits a high resolution of all the photosynthetic pigments of leaf tissues within 13 min.

Experimental Plant material. Control and iron-deficient maize plants (Zea mays L). were cultured hydroponically in Hoagland's nutrient solution as previously described (11,12).

The plants were grown in a controlled environment chamber (photosynthetically-active at a photon flux density, PPFD, 250 µmol m-2 s-1, 24oC, 80% relative humidity and a 16 h photoperiod). Iron-deficient plants were obtained by removing Fe from the culture solution, adding 100 g of CaCO3, and adjusting the pH to 8 with NaOH. To induce the formation of zeaxanthin by the de-epoxidation of violaxanthin, iron deficient plants were subjected to high light (PPFD 2000 µmol m-2 s-1) for 2 h.

Extraction procedures. Photosynthetic pigments were extracted from control and iron-deficient leaf discs by grinding in a mortar with 10 mL of cold acetone, to which a few mg of sodium ascorbate were added to avoid phaeophytin formation. During pigment extraction, all extraction procedures were carried out in dim room light (less than 8 µmol m-2 s-1). Chromatography. The method was developed initially with a Beckman Gold System equipped with a 4 µm particle diameter, 25 cm steel column with ODS reversed phase C18 using a flow rate of 1.5 mL min-1. The method was then modified for a simple modular system consisting of a single pump (Waters M-51) operating at a flow rate of 2 mL min-1, a Rheodyne 7010 injector with a 20 µL loop, a 10 cm long, 4 µm particle diameter, radial compression rp-C18 column (Nova-pack,

Waters).

Pigments

were

detected

at

440

nm

with

a

spectrophotometric detector (Shimadzu SPD-6AV) and the peak areas integrated with a PC Integration Pack (Version 1.0; Softron). All solvents were HPLC grade. Identification of pigments. The identification of each fraction of the pigment extracts was carried out by UV-Visible spectrophotometry (Shimadzu UV-3000) following a method previously described (13,11) with minor modifications. Each fraction was collected from the detector, then diluted with 2 mL of water in order to increase the polarity, and then passed through a C18 Sep-Pack (Waters). The fraction retained in the cartridge was then eluted with ethyl ether and evaporated in the dark using nitrogen. The crystalline residue was dissolved in either carbon disulphide, ethanol or hexane and the spectra were determined over 350-550 nm. The purity of the pigments was monitored by HPLC analysis.

Results The mobile phase developed in this method is based on a dual isocratic step. The starting phase is a mixture of 1.75% methanol, 1.75% dichloromethane,

1.75% water and 94.75% acetonitrile and the second is 50% acetonitrile and 50% ethyl acetate. The second phase enters the system 5 min after beginning the chromatography. Figure 1 shows the chromatograms obtained from the pigments extracted from leaves and separated using this HPLC method. A high level of zeaxanthin can be induced in iron-deficient leaves by exposing the leaves to high light intensities (12). Figure 1 demonstrates clearly the large decrease in violaxanthin and appearance of zeaxanthin when iron-deficient leaves are transferred from the dark to the light. It can also be seen that the chromatographic system gives a clear separation of all the major photosynthetic pigments in higher plants, including the pigments from the xanthophyll (violaxanthin) cycle, in approximately 13 min. Quantification of pigments was carried out by chromatography of known amounts of the individual pigments previously isolated. The pigment concentrations were determined by using the extinction coefficients reported previously by (14) for carotenoids and (15) for chlorophylls. The areas under the chromatography peaks were determined and the conversion factors between the area and pigment concentration calculated for each pigment (Table I). The degree of separation between two adjacent compounds, designated i and

j for the first and second respectively, in a chromatographic column is

defined by:

tj − ti Ri , j = 2 ai + aj

where Ri,j is the power of resolution of the two pigments, ti and tj are the retention times of the components i and j respectively, and ai and aj are the base width of the two peaks associated i and j (16). The results obtained by applying this formula to the pigment peaks from the extracted leaf pigments are given in Table II. A value for Ri,j of 1.5 or greater is generally taken to indicate a good

resolution of two peaks (16). It can be seen from Table II that the Ri,j for zeaxanthin and lutein is 1.77, indicating that a satisfactory separation between these two pigments has been achieved. The absence of overlap between the peaks associated with these two pigments enables integration of the area under the peaks to be made from the baseline in order to quantitate accurately the two pigments. Steel chromatography columns with ODS reversed phase C18 of less than 25 cm in length were found to be unable to resolve satisfactorily lutein from zeaxanthin. Standards of violaxanthin, antheraxanthin and zeaxanthin were obtained from dark adapted and iron-deficient maize leaves. These standards were obtained as described in Materials and Methods. The pigments were dissolved in carbon disulphide, hexane and ethanol and the visible spectra were recorded in the range of 350-550 nm (Figure 2). The most common test for the identification of diepoxy (i.e. violaxanthin) and epoxy (i.e. antheraxanthin) carotenoids, is based on their reaction with hydrochloric acid (14). The reaction involved is the acid-catalyzed isomerization of epoxy-carotenes when a small drop of concentrated hydrochloric acid is added to an ethanolic solution of carotenoid. Applying this procedure to the fractions corresponding to violaxanthin and antheraxanthin, a hypsochromic shift of 17-22 nm for monoepoxides and 40 nm for diepoxides (dotted spectra in Figure 2) can be observed. In routine analyses of pigment extracts from leaves the radial compression rp-C18 columns (Nova-pack, Waters) were found to have a typical operating life span of 600-700 injections. Three of these columns purchased at different times were found to produce results similar to those presented in Figure 1 and Table 1.

Discussion The use of this new chromatographic method to resolve zeaxanthin from lutein facilitates the study of the pigments involved in the xanthophyll cycle.

There can be no doubt from the data presented in Table I of the value of this method for the separation of photosynthetic pigments. Even in the case of the isomers, lutein and zeaxanthin, Ri,j (1.77) is well in excess of the acceptable minimum value (1.50). To date there have been no rapid methods reported that give R values for zeaxanthin and lutein higher than 1.2 which are clearly below the minimum required for an acceptable level of resolution between peaks (6,7,8). The recent method of Thayer and Björman (9) reports an Ri,j of 1.56, however this method is considerably more complicated and time consuming than the method reported here. The spectra of the peaks corresponding to violaxanthin, antheraxanthin and zeaxanthin, shown in Figure 2, together with the hypsochromic shift when a drop of HC1 was added demonstrates the identity and purity of the pigments consistent with the literature (14). Iron deficient maize plants were studied because it was shown from previous studies (12) that the xanthophyll cycle was enhanced in iron deficient leaves. After high light treatment of these plants, the area under the peak corresponding to zeaxanthin was similar to that of lutein, no antheraxanthin could be detected, and most of violaxanthin was converted to zeaxanthin (Figure 1). This clearly is a useful situation to evaluate the effects of overlap between these peaks. It is also interesting to observe the small decline in the baseline after the end of the peak corresponding to zeaxanthin. This is due to absorbance changes when the second phase enters the system (Figure 1) and it is more clearly observed when the sample injected is more dilute, as is the case of extractions from iron deficient leaves which can contain pigment concentrations less than 10% of that found in the extracts from control leaves (12). The spectra of the peaks corresponding to violaxanthin, antheraxanthin and zeaxanthin, shown in Figure 2, together with the hypsochromic shift when a drop

of HCl was added demonstrated that the identity and purity of the pigments resolved by this chromatographic method are consistent with standards previously reported in the literature (14). Although the method reported in this paper simplifies the mobile phase and the gradient used compared to previously published methods, the two main advantages of the method are its speed and the use of basic HPLC equipment with only a single pump.

Consequently, this

method can be readily used on any low cost isocratic HPLC equipment to provide a rapid and accurate procedure for pigment analysis. No special care has to be taken in the preparation of the mobile phase and no degradation of the samples has been observed.

References

1. B. Demmig-Adams. Carotenoids and photoprotection in plants: a role for the xanthophyll zeaxanthin. Biochim. Biophys. Acta, 1020: 1-34. (1990) 2. A.

Hager.

Die

zusammenhänge

zwischen

lichtinduzierten

xanthophyll-umwand-lungen und Hill-reaktion . Ber. Deutsch. Bot. Ges, 79: 94-107. (1966) 3. D. Siefermann and H. Yamamoto. Light-induced de-epoxidation of violaxanthin in lettuce chloroplasts. IV. The effect of electron transport conditions on violaxanthin availability. Biochim. Biophys. Acta, 387: 149-158. (1975) 4. A.

Hager.

Lichtbedingte

chloroplasten-kompartiment

als

violaxathin--zeaxanthin-umwandlung;

pH-erniedrigung ursache

der beziehungen

photophosphorylierung. Planta, 89: 224-243. (1969)

in

einem

enzymatishen zur

5. A. Hager. The reversible, light-induced conversions of xanthophylls in the chloroplast. In Pigments in Plants. F.C. Czygan. eds. Fisher, Stuttgart-New York, 1980. pp. 57-59 6. R.K. Juhler and R.P. Cox. High-performance liquid chromatographic determination of chloroplasts pigments with optimized separation of lutein and zeaxanthin. J. Chromatogr, 508: 232-235. (1990) 7. J. Rivas, A. Abadía and J. Abadía. A new reversed phase-HPLC method resolving all major higher plant photosynthetic pigments. Plant Physiol, 91: 190-192. (1989) 8. D.

Siefermann-Harms.

High

performance

liquid

chromatography

of

chloroplast pigments. One-step separation of carotene and xanthophyll isomers, chlorophylls and phaeophytins. J. Chromatogr, 448: 411-416. (1988) 9. S.S. Thayer and O. Björkman. Leaf xanthophyll content and composition in sun and shade determined by HPLC. Photosyn. Res, 23: 331-343. (1990) 10. C. Wilhelm, I. Rudolph and W. Renner. A quantitative method based on HPLC-aided pigment analysis to monitor structure and dynamics of the phytoplankton assemblage - A study from Lake Meerfelder Maar (Eifel, Germany). Arch. Hydrobiol., 1: 21-35. (1991) 11. J. Val, J. Abadía, L. Heras and E. Monge. Higher plant photosynthetic pigment analysis. Determination of carotenoids and chlorophylls. J. Micronutr. Anal, 17: 239-251. (1986) 12. J. Val and E. Monge. Violaxanthin cycle and fluorescence in iron-deficient maize leaves. Curr Res Photosyn, 4: 765-768. (1990) 13. E. Monge, J. Val and L. Heras. Identificación de pigmentos en plantas superiores por cromatografía líquida en fase reversa. An Aula Dei, 17: 33-43. (1984)

14. B.H. Davies. Carotenoids. In Chemistry and Biochemistry of Plant Pigments. T.W. Goodwin. eds. Academic Press, London, 1976. Vol 2. pp. 38-165 15. J.F.G.M. Wintermans and A.D. Mots. Spectrophotometric characteristics of chlorophylls a y b and their phaeophytins in ethanol. Biochim Biophys Acta, 109: 448-453. (1965) 16. J.M. Storch. Fundamentos de la cromatografía de gases. Editorial Alhambra. Madrid, 1975.

Table I. Retention time (ti), width at the base of the peak (ai),resolution between adjacent pigments (Ri,j) and concentration (Conc.) of the photosynthetic pigments contained in an extract of iron deficient maize leaves that have been exposed to a high light treatment. Means and standard deviation (in brackets) and coefficient of variation (CV) of ten chromatograms of the same sample are given Pigment Neoxanthin Violaxanthin Antheraxanthin Lutein Zeaxanthin Chlorophyll b Chlorophyll a ß-carotene

ti (min)

CV

ai (min)

CV

2.03 (0.03) 3.06 (0.04) 4.58 (0.08) 6.87 (0.12) 8.32 (0.16) 9.65 (0.14) 10.39 (0.14) 12.50 (0.16)

1.35 1.34 1.82 1.81 1.87 1.41 1.36 1.30

0.73 (0.02) 0.48 (0.02) 0.44 (0.03) 0.81 (0.01) 0.81 (0.02) 0.33 (0.01) 0.41 (0.01) 0.84 (0.03)

2.91 3.31 7.23 1.71 1.92 2.91 1.96 3.23

Ri,j -1.69 (0.14) 3.28 (0.28) 3.64 (0.25) 1.77 (0.08) 2.32 (0.12) 2.00 (0.11) 4.75 (0.21)

CV -8.75 8.59 6.98 4.60 5.12 5.42 4.49

Conc. (nmol cm-2) 0.34 (0.01) 0.08 (0.00) 0.08 (0.00) 1.76 (0.06) 0.70 (0.03) 2.03 (0.05) 9.30 (0.22) 0.93 (0.04)

CV 1.58 3.83 5.83 3.29 4.13 2.38 2.32 4.25

Table II.- Conversion factors and extinction (C.F.) coefficients ( E quantification of pigments. Pigment Neoxanthin Violaxanthin Antheraxanthin Lutein Zeaxanthin Chlorophyll b Chlorophyll a ß-carotene

C.F. 1.55 x 10-5 1.42 x 10-5 1.44 x 10-5 1.78 x 10-5 1.81 x 10-5 2.57 x 10-5 3.62 x 10-5 2.03 x 10-5

E

% 1cm

2243 2550 2800 2550 2540 520 918 2620

% 1cm )

λ(nm)

Solvent

439 443 442 445 450 645 662 453

Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol

for the

The coefficients of extinction were reported by Davies (14) for carotenoids and Wintermanns and De Mots (15) for chlorophylls.

Figures

Figure 1. HPLC traces of pigment separations of pigments extracted from iron deficient maize leaves. Left chromatogram corresponds to leaves dark adapted overnight and right chromatogram is for similar leaves that had been exposed for two hours to high light treatment. The identity of the pigment fractions is: 1, Neoxanthin; 2, violaxanthin; 3, Antheraxanthin; 4, Lutein; 5, Zeaxanthin; 6, Chlorophyll b; 7, Chlorophyll a; and 8, ß-Carotene

Figure 2. Spectra of the xanthophyll cycle pigments purified by this chromatographic procedure. Spectra are shown for the pigments solvated in (A) carbon sulfide, (B) hexane, (C) ethanol. The dotted line indicates the acid-induced shift corresponding to de-epoxidation of the pigments. The absorption maxima for the peaks shown here correspond with ±1 nm to those reported previously by Davies (14).