Radiolabeling of Chlorophyll by [14C]Glutamic Acid in vivo and Relative Quantification of Labeled Chlorophyll by Using Thin Layer Chromatography (TLC)

Materials and Reagents

1. Synechocystis PCC 6803
   
2. Glutamic acid [U-C14] (ARC 0165A, American Radiolabeled Chemicals) ([¹⁴C]Glu)
3. Methanol
4. 25% ammonia solution
   
5. 1 M NaCl
6. Hexane
7. 10% KOH
8. Petroleum ether
9. Chloroform
10. 1 M Na₂HPO₄
11. 1 M NaH₂PO₄
12. X-ray film (Eastman Kodak Company)
13. NH₄OH
    
14. 1 M TES [2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid]/NaOH (pH 8.2)
15. Growth medium BG11

Equipment

1. 10 ml Headspace vials (Sigma-Aldrich)
2. Water bath shaker
3. 2 ml o-ring cap tubes
   
4. Glass beads (100-200 μm)
   
5. Vortex
6. Tabletop centrifuge (MiniSpin plus, Eppendorf)
7. Speedvac Concentrator plus (Eppendorf)
8. Silica gel TLC plate (SIL G-25, MACHEREY-NAGEL)
9. Rectangular TLC developing tank (Sigma-Aldrich)
10. Mikro 22R centrifuge (Hettich)
11. MiniSpin centrifuge (Eppendorf)

Procedure

1. Labeling requires a 25-ml culture of cyanobacterial cells at the exponential phase of growth. Protocol works very well for the cyanobacterium Synechocystis PCC 6803 grown at 30 °C in growth medium BG11 (Rippka et al., 1979) to optical density at 750 nm~0.4. Harvest cells by centrifuging at 3,500 x g for 5 min at room temperature using brake 5 (Mikro 22R centrifuge).
2. Discard supernatant and resuspend cell pellet in 2 ml of growth medium BG11, supplemented by 20 mM TES (pH 8.2). Transfer cells into a 2 ml tube and centrifuge at 3,500 x g for 2 min at room temperature (MiniSpin centrifuge).
3. Discard supernatant and resuspend cell pellet in 450 μl of growth medium/20 mM TES. Transfer cells into a glass vial.
4. Incubate cells in a water bath shaker for 30 min at 100 rpm under light and temperature conditions you prefer for your experiment.
5. Add 180 μM of [¹⁴C]Glu dissolved in growth medium. Incubate under the same conditions for another 30 min.
6. Transfer the labeled culture into a 2 ml o-ring cap tube. Spin down the cells at 7,000 x g for 2 min at room temperature and discard supernatant. At this point, the cell pellet can be frozen in liquid nitrogen and stored at -70 °C or used immediately for following pigment extraction.
7. Resuspend cells in 1 ml of H₂O and pellet cells at 7,000 x g for 2 min at room temperature. Discard water and repeat wash twice by 1 ml of H₂O to remove all traced of labeled [¹⁴C]Glu. Resuspend cells in 1 ml of methanol/0.2% NH₄OH. Add 50 μl of glass beads and vortex for 1 min to facilitate pigment extraction. Spin down 4 min at max rpm. Work under a dim light for all following steps
8. Transfer supernatant (~1 ml) into 2 ml tube and add another 300 μl of methanol/0.2% NH₄OH to cells, vortex and spin down again. Combine supernatants and add 140 μl of 1 M NaCl.
9. Add 400 μl of hexane, vortex and spin down 30 sec at max rpm to accelerate phase separation. Collect upper phase containing chlorophyll. Repeat step 9 three times and combine all hexane into a 2 ml tube.
10. Evaporate hexane using SpeedVac concentrator set to V-AL and 30 °C for 5 min.
11. Resuspend the pellet in 190 μl of methanol and add 10 μl of 10% KOH. Incubate at room temperature for 15 min to convert chlorophyll into phytol-less Mg-chlorin.
12. Extract this solution by 200 μl of hexane and discard upper phase, repeat 4-times.
13. Transfer remaining ~150 μl of the methanol phase into a new 0.5 ml tube. Evaporate this solution using a SpeedVac concentrator to final volume of 30-50 μl.
14. Extract this solution 5 times by 150 μl of petroleum ether and discard upper phase containing carotenoids.
15. Evaporate completely at 30 °C for 30 min and resuspend pellet in 30 μl of methanol:chloroform (1:1). Load 10 μl on the TLC plate. 5 μl can be used for measurement using scintillation counter (see next step).
16. Load 10 μl of pigment solution on a silica gel TLC plate. Place the plate in a TLC developing tank with 300 ml of mobile phase – methanol: 10 mM Na_XH_YPO₄, pH 6.8 (3:1, v/v). Remove the plate from the tank after 30 min of incubation and dry it at 37 °C for 10 min. Expose the plate to X-ray film for 3-5 days and develop. After development only a one signal should be detected corresponding to Mg-chlorin ‘green’ band on TLC.
    Note: An alternative to TLC followed by detection on an X-ray film is to use a scintillation counter and measure directly radioactivity in the final pigment fraction (5 μl). The later method is faster; however it is less sensitive and also accurate due to presence of ¹⁴C labeled impurities.
    

Acknowledgments

We acknowledge the financial support from the project Algatech (CZ.1.05/2.1.00/03.0110) and Czech Science Foundation, project no. P506/12/1522.

References

1. Koreny, L., Sobotka, R., Janouskovec, J., Keeling, P. J. and Obornik, M. (2011). Tetrapyrrole synthesis of photosynthetic chromerids is likely homologous to the unusual pathway of apicomplexan parasites. Plant Cell 23(9): 3454-3462.
   
2. Rippka,R., Deruelles, J., Waterbury, J. B., Herdman, M. and Stanier, R. Y. (1979). Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J Gen Microbiol 111(1): 51-61.