Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis

Podsumowanie

Here, we present a protocol to quantitatively determine phycobiliprotein content in the cyanobacterium Synechocystis using a spectrophotometric method. The extraction procedure was also successfully applied to other cyanobacteria and algae strains; however, due to variations in pigment absorption spectra, it is necessary to test the spectrophotometric equations for each strain individually.

Streszczenie

This is a simple protocol for the quantitative determination of phycobiliprotein content in the model cyanobacterium Synechocystis. Phycobiliproteins are the most important components of phycobilisomes, the major light-harvesting antennae in cyanobacteria and several algae taxa. The phycobilisomes of Synechocystis contain two phycobiliproteins: phycocyanin and allophycocyanin. This protocol describes a simple, efficient, and reliable method for the quantitative determination of both phycocyanin and allophycocyanin in this model cyanobacterium. We compared several methods of phycobiliprotein extraction and spectrophotometric quantification. The extraction procedure as described in this protocol was also successfully applied to other cyanobacteria strains such as Cyanothece sp., Synechococcuselongatus, Spirulina sp., Arthrospira sp., and Nostoc sp., as well as to red algae Porphyridium cruentum. However, the extinction coefficients of specific phycobiliproteins from various taxa can differ and it is, therefore, recommended to validate the spectrophotometric quantification method for every single strain individually. The protocol requires little time and can be performed in any standard life science laboratory since it requires only standard equipment.

Wprowadzenie

fPhycobiliproteins are water-soluble pigment-protein complexes that represent major components of the light-harvesting antennae in prokaryotic cyanobacteria (Cyanophyta) and several eukaryotic taxa (Glaucophyta, Rhodophyta, and Cryptophyta)¹. They occur mainly as supramolecular complexes called phycobilisomes and they are typically attached to the surface of the photosynthetic membranes on the stromal side, with the exception of Cryptophyta, where the phycobiliproteins are localized in the thylakoid lumen². Four types of phycobiliproteins have been identified up to date: the core allophycocyanin, and the peripheral phycocyanin, phycoerythrin, and phycoerythrocyanin¹. As the main light-harvesting complexes, phycobilisomes represent one of the crucial factors of algae and cyanobacteria mass cultures productivity. It has been demonstrated that phycobilisomes truncation can enhance biomass accumulation under strong light³. On the other hand, under modest or low irradiance, the antenna truncation resulted in growth rates and biomass accumulation reduction^3,4. Phycobiliproteins are commercially used as food colorants, pharmaceuticals, and food additives, in the cosmetic industry, and as fluorescence probes with applications in flow cytometry, fluorescent immunoassays, and fluorescence microscopy⁵.

This protocol focuses on the quantitative determination of phycobiliproteins in the model cyanobacterium Synechocystis. Cyanobacteria are the earliest oxygenic photosynthetic autotrophs; they have been forming the Earth's biosphere for more than 2.4 billion years⁶. They play a crucial role in global biogeochemical cycles of nitrogen, carbon, oxygen, and other elements. Among cyanobacteria, a unicellular strain Synechocystis gained a unique position since it was the first cyanobacterium with the entire genome sequenced^7,8, it is naturally transformable by exogenous DNA⁹, and it performs stable and relatively fast growth^10,11. In Synechocystis, the core antenna component, allophycocyanin, is associated with the integral membrane proteins, and the attached phycocyanin is located on the thylakoid membrane periphery.

Several methods for phycobiliprotein extraction and quantification are compared within this protocol. The final extraction procedure was successfully applied to Synechocystis, as well as to other cyanobacteria strains, including Cyanothece sp., Synechococcuselongatus, Spirulina sp., Arthrospira sp., and Nostoc sp., and it was also successfully applied to red algae Porphyridium cruentum. Therefore, the method developed in this protocol can be considered as a universal method for phycobiliprotein extraction. Even though some of the tested extraction methods resulted in higher total protein yields, the here described extraction procedure provided the highest phycobiliprotein yields together with the lowest content of chlorophyll a residue in the phycobiliprotein extract. Reducing the content of chlorophyll a was essential for the correct phycocyanin and allophycocyanin spectrophotometric quantification.

The phycobiliprotein absorption spectra can vary significantly among various algae and cyanobacteria species^{12,13,14,15,16,17} and even among several strains of a single cyanobacteria genus¹⁸. Therefore, the specific wavelengths and absorption coefficients as used for the determination of phycocyanin and allophycocyanin in Synechocystis are not generally applicable to other strains. Additionally, Synechocystis does not contain phycoerythrin and phycoerythrocyanin that can be found in some other algae and cyanobacteria. For the purpose of the determination of phycobiliproteins in strains other than Synechocystis, it is recommended to evaluate the spectrophotometric equations for each strain individually.

Although the protocol contains two longer steps (overnight freeze-drying of the cellular pellets and 1-hour protein extraction), the total labor time for the phycobiliproteins quantification is no longer than 2 hours.

Protokół

1. Cyanobacteria Cultivation

1. Cultivate Synechocystis cells in Erlenmeyer flasks or in photobioreactors^10,19 in buffered BG11 medium²⁰ to maintain a pH of < 10 (e.g., using 17 mM HEPES¹⁰).
   NOTE: Standard cultivation conditions require a controlled temperature (typically, 30 °C, the optimal temperature is 35 °C)²¹, illumination (typically, a white light of an intensity up to 800 µmol[photons]/[m²·s])²¹, and a CO₂ supply (in the 400-mL flat-panel photobioreactor, the growth saturating CO₂ concentration is 1,700 ppm)²¹.
2. To determine the culture density, measure the culture optical density spectrophotometrically at 730 nm (OD₇₃₀) using a cuvette with a light path of 1 cm. Alternatively, count the cells under a microscope using a hemocytometer or an automated cell counter.

2. Samples Preparation

1. Work under low irradiance to prevent phycobiliprotein degradation.
2. Harvest 3 x 1 mL of culture suspension to safe-lock tubes. Use triplicates for the estimation of technical errors in the measurement. To perform the culture sampling under sterile conditions, harvest the cells in a laminar hood and follow the proper working and safety practices.
3. Centrifuge the cells at 15,000 x g at laboratory temperature for 5 min. Pay attention to the properly balanced centrifuge rotor. After centrifugation, discard the supernatants. Be sure not to disturb the pellet.
4. Put the samples in a freezer. For long-term storage, keep the samples at -80 °C. This is necessary to prevent phycobiliprotein degradation. For short-term storage, -20 °C is sufficient.
5. Freeze-dry the samples overnight. For a proper freeze-drying process, keep the temperature of the freeze-dryer condenser below -60 °C and the pressure in the freeze-dryer chamber around 1 hPa.
6. After finishing the freeze-drying cycle, close the tube as soon as possible to prevent the reabsorption of water from the air.

3. Cell Homogenization and Pigments Extraction

1. Add four pieces of glass beads (with a diameter of 2 mm) to each sample tube and close the tubes.
   NOTE: When the lid of the safe-lock tube used for homogenization is too thin, it can break during the homogenization; therefore, only safe-lock tubes with strong lids are recommended for this protocol.
2. Homogenize the samples with the glass beads for 15 s on a homogenizer at laboratory temperature.
   NOTE: A properly homogenized sample is spread over the whole inner surface of the safe-lock tubes.
3. Add 1 mL of PBS buffer (pH 7.4), precooled to 4 °C, to the samples in order to extract phycobiliproteins.
   NOTE: From this step on, keep the samples on ice to prevent the degradation of the extracted proteins. This is critical.
4. Mix the samples with PBS for 5 s on the homogenizer at laboratory temperature.
   NOTE: After mixing, the samples are greenish.
5. After mixing, keep the samples on ice for 60 min. Cover the ice bath with a lid to prevent pigments degradation.
6. After 60 min of phycobiliprotein extraction, centrifuge the samples at 15,000 x g at 4 °C for 5 min. Pay attention to properly balance the centrifuge rotor.
   NOTE: After the centrifugation, the supernatant has a cyan blue color.

4. Phycobiliprotein Quantification

1. Before the spectrophotometric measurement, calibrate the spectrophotometer to the baseline using the PBS buffer as a blank.
2. Once the spectrophotometer is calibrated, discard the PBS from one spectrophotometer cuvette and pipette the supernatant with extracted phycobiliproteins instead of with the discarded buffer.
3. Quantify the phycobiliprotein concentration spectrophotometrically, using a slit width of 0.5 nm.
   1. Measure the absorbance of the phycocyanobilins in phycocyanin and allophycocyanin against the PBS buffer blank at 615 nm (A₆₁₅) and 652 nm (A₆₅₂), respectively, and measure the absorbance of the cellular debris at 720 nm (A₇₂₀).
   2. Calculate the concentration of phycocyanin and allophycocyanin according to the equations (1) and (2) of Bennett and Bogorad¹²:
      
      
      NOTE: A₆₁₅, A₆₅₂, and A₇₂₀ should fit to the linear absorbance range of the spectrophotometer. If necessary, dilute the sample with PBS buffer.
4. Recalculate the pigment concentration in the original samples. The pigment concentration in the sample corresponds directly with the results of equations (1) and (2) when 1 mL of culture and 1 mL of the extraction buffer are used for the analysis. In case of using different volumes of cyanobacteria samples and/or PBS buffer, the final pigment concentration should be calculated according to equation (3):
      (3)
   NOTE: In case of a normalizing phycobiliprotein content per cell dry weight, the final pigment concentration should be calculated according to equation (4)  (4)

5. Determination of the Cell Dry Weight (Optional)

1. Weigh three empty safe-lock tubes on analytical balances.
   CAUTION: It is critical that the safe-lock tubes are dry. In case of storing the tubes in a wet environment, dry the tubes for several hours in a freeze dryer before weighing them. Manipulate the tubes gently and only with powder-free gloved hands to avoid any contact between the material and the researcher's fingers. Keep all holders and centrifuge clean to avoid the transfer of any material to the tubes.
2. Sample 1 x 15 mL of culture suspension to 15-mL conical tube.
3. Centrifuge the culture suspension in the 15-mL conical tubes at 4,000 x g at laboratory temperature for 10 min. Balance the centrifuge rotor with three additional 15-mL conical tubes, each containing 15 mL of water. After centrifugation, discard 12 mL of the supernatant.
4. Resuspend the pellet in the remaining supernatant and transfer 3 x 1 mL of the mixture to three 1.5-mL safe-lock tubes with a pipette. In case some leftovers of the pellet remain in the 15-mL conical tube, add 1.5 mL of deionized water to the conical tube, vortex or shake the tube to resuspend the remaining pellet, and transfer 3 x 0.5 mL of the mixture to the three 1.5-mL safe-lock tubes (already containing 3 x 1 mL of the pellet) with a pipette.
   NOTE: Dividing the pellet over three 1.5-mL safe-lock tubes will allow the estimation of any technical error of the dry weight measurement.
5. Centrifuge the cells in 1.5-mL safe-lock tubes at 15,000 x g at laboratory temperature for 5 min. Balance the centrifuge rotor with an additional 1.5-mL safe-lock tube that contains 1 mL of water. After the centrifugation, discard the supernatants.
6. Put the samples in the freezer. For long-term storage, keep the samples at -80 °C; for short-term storing, -20 °C is sufficient.
7. Freeze-dry the samples overnight. For a proper freeze-drying process, keep the temperature of the freeze dryer condenser below -90 °C and the pressure in the freeze-dryer chamber around 1 hPa.
8. After freeze-drying, close the tubes and weigh the samples on analytical balances.
   NOTE: The dry weight value can be used for the normalization of the phycobiliprotein content per cell dry weight.

Wyniki

For the initial method tests, Synechocystis was cultivated as batch cultures in Erlenmeyer flasks on a shaker in BG11 cultivation medium²⁰ (supplemented with 17 mM HEPES) at 25 °C, under a warm white light of an intensity of 50 µmol(photons)/(m²·s) and with 1% CO₂ in the culturing atmosphere. During the cultivation, the cultures were sampled to safe-lock tubes and centrifuged (15,000 x g at laboratory temperatu...

Dyskusje

This protocol describes a simple, fast, and reproducible method for the quantification of phycobiliprotein content in the model cyanobacterium Synechocystis. Several methods of cell homogenization, protein extraction, and phycocyanin and allophycocyanin quantification are compared, and the final protocol represents a combination of the optimal steps of every single procedure. As representative data, the content of phycobiliproteins was quantified in Synechocystis cells under increasing light intensity. ...

Materiały

Name	Company	Catalog Number	Comments
Synechocystis sp. PCC 6803	Institut Pasteur, Paris, France	6803	Cyanobacterium strain
Roti-CELL PBS	Carl Roth GmbH + Co. KG, Karlsruhe, Germany	9143.1	Phosphate-Buffered Saline (PBS) solution, pH 7.4
Eppendorf safe-lock tubes	Eppendorf, Hamburk, Germany	30120086	Safe-lock tubes 1.5 ml
VWR 80-Place Storage System	VWR International, Radnor, Pennsylvania, USA	30128-282	Holder for safe-lock tubes
RAININ 100 µl -1000 µl	Mettler-Toledo, Columbus, Ohio, USA	17014382	Pipette
GP-LTS-A-1000µL-/F-768/8	Mettler-Toledo, Columbus, Ohio, USA	30389272	Pipette tips
Rotina 420R	Hettich, Kirchlengern, Germany	4701	Refrigerated centrifuge for 1.5 ml safe-lock tubes and 15 ml conical centrifuge tubes
LCexv 4010	Liebherr, Bulle, Switzerland	9005382197172	Refrigerator and freezer -20 °C
Revco ExF -86°C Upright Ultra-Low Temperature Freezer	Thermo Fisher Scientific, Waltham, Massachusetts, USA	EXF24086V	Freezer -80 °C
CoolSafe	LaboGene, Lillerød, Denmark	7.001.000.615	Freeze dryer
UV-2600	Shimadzu, Kyoto, Japan	UV-2600	Spectrophotometer
Hellma absorption cuvettes, semi Micro	Sigma-Aldrich, St. Louis, Missouri, USA	Z600288	VIS/UV-VIS semi-micro cuvettes 0.75-1.5 ml, spectral range 200-2500 nm
Silamat S6	Ivoclar Vivadent, Schaan, Liechtenstein	602286WU	Homogenizer
Solid-glass beads	Sigma-Aldrich, St. Louis, Missouri, USA	Z273627	Glass bead of the diameter 2 mm
CPA225D-0CE	Sartorius AG, Göttingen, Germany	SECURA225D-1OBR	Analytical balances
C-Phycocyanin from Spirulina sp.	Sigma-Aldrich, St. Louis, Missouri, USA	P2172	Phycocyanin standard
Allophycocyanin	Sigma-Aldrich, St. Louis, Missouri, USA	A7472	Allophycocyanin standard
Bicinchoninic Acid Kit	Sigma-Aldrich, St. Louis, Missouri, USA	BCA1, B9643	Complete kit for total proteins determination
AlgaeTron	Photon System Instruments Ltd., Drásov, Czech Republic	AG 130-ECO	Cultivation chamber for E. flasks, with controllable light and atmosphere
Photobioreactor	Photon System Instruments Ltd., Drásov, Czech Republic	FMT-150	Cultivation equipment for cyanobacteria and algae with completely controllable environment
Cellometer	Nexcelom Bioscience, Lawrence, Massachusetts, USA	Auto M10	Cell counter
Corning 15 mL centrifuge tubes	Sigma-Aldrich, St. Louis, Missouri, USA	CLS430791	15 ml Centrifuge tube for dry weigth sampling
Herasafe KS	Thermo Fisher Scientific, Waltham, Massachusetts, USA	51024579	Laminar flow hood