Analysis of pheophorbide a and other intermediates of the chlorophyll
metabolism in Arabidopsis leaves by HPLC
Ryouichi Tanaka
Inst Low Temp Sci, Hokkaido Univ, N19 W8, Kita-ku, Sapporo, 060-0819, JAPANrtanaka@lowtem.hokudai.ac.jp
SummaryThis article provides a quick guide for the analysis of various intermediates of thechlorophyll metabolism by HPLC. The method is based on that developed by Zapata et al(Zapata et al., 2000) which uses a C8 column for the separation of chlorophylls andcarotenoids. Our lab protocol incorporates a slight modification of the original method forthe analysis of various chlorophyll intermediates in the leaves of higher plants.
Introduction
Chlorophyll is synthesized from glutamate and degraded to several different monopyrrole
molecules in higher plants (Hortensteiner, 2006; Tanaka and Tanaka, 2007).

Some ofthe intermediates of chlorophyll metabolism play essential and multiple roles in various
aspects of the cellular activities in higher plants.

Readers may refer to the recent review

articles on the biosynthetic and degradation pathways for chlorophyll and on the roles of
the tetrapyrroles in plants (Hortensteiner, 2006; Nott et al., 2006; Tanaka and Tanaka,2007).

In this article, I describe an HPLC method used in our laboratory to analyze

chlorophyll precursors and catabolites. We slightly modified the method originallydeveloped by Zapata et al (Zapata et al, 2000) in order to shorten the overall analysis time
without sacrificing the resolution. The original article explained that several carotenoid
species can be separated by the method described; we, however, haven’t yet determined if
this method can be used for the accurate quantification of carotenoids. Specifically, wesuspected that some portions of violaxanthin and neoxanthin could be isomerized. For an
HPLC analysis, experience in dealing with an HPLC system may be required.
Researchers who have never learned basic HPLC techniques may wish to consult withmore experienced colleagues. A Japanese version of this guide is available on
http://www.lowtem.hokudai.ac.jp/plantadapt/ayumi/.
Materials

Standard pigments may be required for quantification purposes. Below is a list of possible
sources for several different pigments.
Chlorophyll a and b: Juntec Co. Ltd, Odawara, Japan
Hydroxymethyl chlorophyll a: Prepared from chlorophyll b according to the methodof Ito et al., (Ito et al., 1996).
Pheophorbide a: Wako Chemicals, Tokyo, Japan
Protoporphyrin IX, Mg-Proto IX, Mg-Proto IX monomethylester: Frontier
Scientific, UT, USA, http://www.frontiersci.com/
Carotenoids: DHI, Hørsholm, Denmark, http://www.dhigroup.com/
The absorption coefficients of various photosynthetic pigments are summarized in thereference by Rebeiz (2002).
An HPLC system with which two solvents can be run is required (Tanaka et al., 2003;
Nagata et al., 2005).
Column: Symmetry C8 (150 x 4.6 mm, 3.5 µm particle size. Waters)
Pump and mixer (A low pressure gradient system is sufficient.)
Detector: Choose either a UV-VIS detector, a photodiode array (PDA) detector, or afluorescent detector. A PDA detector is the best choice for identifying pigments, while afluorescent detector is the best option for sensitivity. UV-VIS detectors are the mosteconomical.
Guard column: Phenomenex AJO-4290 C8 4.0 L mm x 3.0 ID mm with a guardcolumn holder, Phenomenex KJO-4282
For grinding many leaves at a time, we use a special grinding apparatus known as aShakeMaster (BioMedical Science Co. Ltd., Tokyo, Japan). For the extraction ofpigments from a few samples we use a glass homogenizer available from AsOne, Tokyo,Japan, or simply a pestle and mortar.
Extraction of pigments(1) Weigh and grind leaves in acetone using a ShakeMaster grinding apparatus(BioMedical Science Co. Ltd., Tokyo, Japan).

Note: We typically use more than 200 µl of acetone to extract pigments from 10-20 mgof fresh or frozen leaves. It is important to use a sufficient amount of acetone because if
an insufficient amount of acetone is used a substantial portion of chlorophyll will be
converted to chlorophyllide by the action of chlorophyllase present in leaves.
After extraction, minimize exposure of extracts to light.
(2) Centrifuge extracts for 5 min at 10,000 g.
(3) Collect supernatant in a new tube. Repeat extraction with acetone and combine thesupernatants.
(4) Add water so that the final concentration of acetone is 80%.
Note: A portion of the chlorophyll will be gradually epimerazed in acetone. Roughly
speaking, 10% of chlorophyll a could be modified to form a chlorophyll a allomer
within 24 h, which compound typically comes over a minute earlier than chlorophyll ain the HPLC system described in this guide. Moreover, the majority of β-carotene may
be precipitated within 24 h.
(5) Load an aliquot of extracts immediately to the HPLC system.
HPLC analysis(1) You can load an aliquot from your samples directly to the HPLC system. Theinjection volume is typically from 10 µl to 200 µl, depending on the maximum volume
that can be injected to your HPLC system.
(2) We use the following solvents:
A: Methanol:Acetonitrile:0.25M aqueous pyridine = 50:25:25 (v:v:v)
B: Methanol:acetonitrile:acetone = 20:60:20 (v:v:v)(3) The flow rate is 1.2 ml/min. The time program is as follows:min% (B Sol)0012451798

22.59800(4) Pigments are identified by their spectral patterns and/or co-injection with standards.
Note: Chlorophyllide a and Mg-ProtoIX are eluted at the same time. These pigments
should be identified with a photodiode array detector.
(5) Pigments are quantified by the integrated area of each pigment at a certain
wavelength according to the calibration curve made with standard pigments. Most
photosynthetic pigments including carotenoids can be detected at 410 nm.
Chlorophyll precursors can be specifically monitored at 650 nm. For fluorometricdetection of Mg-ProtoIX and Mg-ProtoIX ME, we use an excitation at 417 nm anddetect fluorescence at 600 nm.

References
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Ito H, Ohtsuka T, Tanaka A (1996) Conversion of chlorophyll b to chlorophyll a via
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Nagata N, Tanaka R, Satoh S, Tanaka A (2005) Identification of a Vinyl Reductase
Gene for Chlorophyll Synthesis in Arabidopsis thaliana and Implications for theEvolution of Prochlorococcus Species. Plant Cell 17: 233-240
Nott A, Jung HS, Koussevitzky S, Chory J (2006) Plastid-to-nucleus retrogradesignaling. Annu Rev Plant Biol 57: 739-759
Rebeiz CA (2002) Analysis of intermediates and end products of the chlorohyll
biosynthetic pathway. In AG Smith, M Witty, eds, Heme, Chlorophyll, and Bilins:
Methods and Protocols. Humana Press, NJ, pp 111-155
Tanaka R, Hirashima M, Satoh S, Tanaka A (2003) The Arabidopsis-accelerated cell
death gene ACD1 is involved in oxygenation of pheophorbide a: Inhibition of
the pheophorbide a oxygenase activity does not lead to the "stay-green"
phenotype in Arabidopsis. Plant & Cell Physiology 44: 1266-1274

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