Photosynthesis Research 52: 69±73, 1997. 69
c 1997 Kluwer Academic Publishers. Printed in the Netherlands.
Technical communication
A routine method to determine the chlorophyll a, pheophytin a and
-carotene contents of isolated Photosystem II reaction center complexes
Camiel Eijckelhoff & Jan P. Dekker Department of Physics and Astronomy, Institute of Molecular Biological Sciences, Vrije Universiteit, De
Boelelaan 1081, 1081 HV Amsterdam, The Netherlands; Author for correspondence
Received 13 February 1997; accepted in revised form 28 March 1997
Key words: chlorophyll, pheophytin, Photosystem II, reaction center
Abstract
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 coef®cients of the puri®ed 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.
Abbreviations: Car ± carotene; Chl ± chlorophyll; HPLC ± high-performance liquid chromatography; Pheo ± pheophytin; PS ± Photosystem; RC ± reaction center
Introduction content of the PS II RC complex, since the number and energies of bound chlorophylls seriously affect the The smallest unit of Photosystem II (PS II) capable of interpretations of functional studies of the PS II reac- primary charge separation is the D1±D2-cytochrome tion center, such as those on the primary energy and b-559 reaction center complex (Nanba and Satoh electron transfer reactions (van Gorkom 1995). 1987). This complex is structurally related to the well- In a previous report we analyzed several HPLC characterized RC complex of photosynthetic purple and spectroscopic methods for the determination of
bacteria and binds Chl a,Pheoaand -Car molecules the pigment stoichiometry of the PS II RC (Eijckelhoff as co-factors involved in the primary energy and elec- and Dekker 1995) and concluded that in some reports tron transfer reactions. Despite intensive research, the the Chl to Pheo ratio has been underestimated to very exact number of bound pigment molecules per com- signi®cant extents. In particular some of the applied plex has for long been a matter of debate. Kobayashi spectroscopic methods were suggested to yield rather et al. (1990) and Gounaris et al. (1990) suggested that unreliable results. This is unfortunate, because gener- the PS II RC complex binds 6 Chl a,2Pheoaand ally spectroscopic methods are well-suited for routine
2 -Car molecules. Other groups, however, suggested analysis of PS II RC preparations (they are easy to that the PS II RC complex in its most pure form binds perform and do not require special equipment). An
4Chla,2Pheoaand 1 -Car molecules (Montoya additional advantage of spectroscopic methods is the
et al. 1991) and that higher contents of Chl a and - relative ease by which PS II RC complexes prepared Car arise from contamination with the core antenna in different laboratories can be compared. protein CP47 (Chang et al. 1994; Pueyo et al. 1995). In this report, we describe a very simple spectro- It is important to solve the question of the pigment scopic method well-suited for routine analysis to deter-
PIPS NO.:138356 (M) BIO2KAP
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mine the Chl a,Pheoaand -Car contents of PS II RC preparations from the room temperature absorption spectra of their extracts in 80% acetone. The results are in line with the conclusion of a number of recent detailed studies (Eijckelhoff et al. 1996; Zheleva et al. 1996; Kurreck et al. 1997) that the isolated PS II RC complex in its most stable and pure form binds 6 Chl
a,2Pheoaand 2 -Car molecules.
Experimental
The aim of this work is to determine the concentrations
of Chl a, Pheo a and -Car of PS II RC preparations
from their extracts in 80% acetone. For each wave- length the absorption A of the extract is given by:
Figure 1. Room temperature absorption spectra of HPLC-puri®ed
A =(c )+ (c )
; ;
chl chl pheo pheo Pheo a (full line) and Pheo a obtained by adding 0.75 mM HCl
c ) +( to puri®ed Chl a (dashed line) in 80% acetone. The spectra were car car; (1) normalized as described in the text.
So, if the molar extinction coef®cients of the three pigments are known at three different wavelengths, the concentrations c of the three pigments in the extract can be determined. Because for some preparations contam- errors in the estimation of the Pheo a extinction coef- ination of Chl b can not always be totally excluded, we ®cients will have relatively large effects on the calcu- present an additional method including this pigment. lated Chl/Pheo ratio of the extract. We prepared Pheo The extraction procedure of PS II RC prepara- a by adding 0.75 mM HCl to puri®ed Chl a in 80% tions from spinach in 80% acetone was described in acetone and recorded the absorption spectrum. Fig- detail before (Eijckelhoff and Dekker 1995). Chl a, ure 1 (dashed line) shows the spectrum of the obtained
Chl b,Pheoaand -Car were obtained from such fraction, which in view of the spectral shape almost
extracts with > 99.9% purity by reversed phase HPLC exclusively consists of Pheo a. The spectrum of HPLC- with 100% methanol as mobile phase (Eijckelhoff and puri®ed Pheo a in 80% acetone is shown as well (Figure Dekker 1995). The peak fractions were collected, dried 1, full line). Both spectra are very similar. Neverthe- by ¯ushing with nitrogen gas and re-solubilized in less, in the spectrum of the acidi®ed fraction a very
80% acetone, after which their absorption spectra were small amount (<1%) of Chl a may be present, because recorded on a Cary 219 spectrophotometer.The spectra the absorption near 430±435 nm is slightly stronger in
of Chl a, Chl b and -Car were normalized to published case of the acidi®ed fraction, whereas the absorption
extinction coef®cients in 80% acetone (86.3 mM 1 near 410±415 nm is slightly reduced. The spectrum