The overall goal of this protocol is to quantitatively determine the phycobiliproteins content in the model cyanobacterium Synechocystis using a spectrophotometric method. Phycobiliproteins are water soluble pigment-protein complexes that represent major components of the light-harvesting antennae in prokaryotic cyanobacteria and several groups of eukaryotic algae. Since they are the main light-harvesting complexes, phycobiliproteins represent one of the crucial factors that determine algae and cyanobacteria productivity.
Synechocystis is a model cyanobacterium which is used in hundreds of laboratories worldwide. Synechocystis contains two phycobiliproteins, phycocyanin and allophycocyanin. This protocol describes simple, efficient and reliable method for quantitative determination of both phycobiliproteins in this mold strain.
We compared several methods, phycobiliproteins extraction and spectrophotometric quantification. The quantification method needs to be tested for every single strain since phycobiliproteins absorption spectra can vary between different strains. On the beginning of the phycobiliproteins extraction, transfer one milliliter of cyanobacterium culture to a safe-lock tube.
If you decide to determine dry weight of the samples, weight the empty tubes before the culture sampling. Be sure that the culture is not sedimented. Centrifuge the cells at 15, 000 G at laboratory temperature for five minutes.
Carefully discard the supernatant. Be sure to not disturb the pellet. Put the samples to freezer.
For long-term storage, keep the samples at 80 degrees Celsius. This is necessary to prevent phycobiliproteins degradation. Once the samples are frozen, insert them into the freeze dryer.
Once the freeze drying has started, check the temperature and pressure on the freeze dryer. The temperature has to be below 60 degrees Celsius and the pressure has to be around one hectare Pascal. Freeze dry the samples overnight.
After finishing the freeze drying cycle, close the tubes as soon as possible to prevent reabsorption of water from air. Add four PCs of two milliliter glass beads to each sample tube. Homogenize the samples with the glass beads for 15 seconds.
Properly homogenized sample is spread over the whole inner surface of the safe-lock tubes. After cells homogenization, add one milliliter of phosphate-buffered saline to the samples in order to extract the phycobiliproteins. Optimal pH of the PBS is around 7.4.
Mix the cells with the PBS for five seconds on the homogenizer. This will secure optimal sample mixing for efficient phycobiliproteins extraction. After mixing, the samples are greenish.
Keep the samples on ice for 60 minutes for the most efficient phycobiliproteins extraction. After the extraction, centrifuge the samples at 15, 000 G and four degrees Celsius for five minutes. After centrifugation, the supernatant has cyan blue color.
Before the spectrophotometric measurement, calibrate the spectrophotometer using the PBS as a blank. Once the spectrophotometer is calibrated discarded PBS from one spectrophotometer cuvette and pipette the supernatant with extracted phycobiliproteins instead of the discarded buffer. For phycocyanin and allophycocyanin quantification the supernatant with extracted phycobiliproteins is measured at 615, 652 and 720 nanometers.
As described in the text, there are several equations for the phycobilisomes quantification known in the literature. The equation used in this protocol was found as the best correlating phycocyanin and allophycocyanin concentrations as the pigments and dots. Critical steps of the protocol are cells homogenization and extraction, that should provide both high yield and high specificity.
We tested several extraction procedures such as cells disruption by sonication, homogenization with zirconia beads, or freeze cell link. The method from this protocol provided the highest phycobilisomes yield. Since even a small contamination of the protein extract by chlorophyll can lead to the phycobiliproteins content overestimation, it is necessary to avoid any chlorophyll extraction from the cells.
We detected chlorophyll in the crude extract, then we used sonication for cells disruption. With repeating sonication cycles, chlorophyll concentration increased. Homogenization time was optimized as 15 seconds.
Homogenization for both five seconds and 20 seconds provided slightly lower yields. Addition of sodium or potassium chloride to the PBS buffer or to water, did not increase phycobiliproteins extraction efficiency, same as extraction in sodium acetate buffer. The phycobiliproteins extraction time was optimized for 60 minutes, since after 120 and even 240 minutes, the phycobiliproteins concentration didn't change significantly.
Make sure that absorbance at 615, 652 and 720 nanometers fit into the near absorbance range of the spectrophotometer. The spectrophotometer used in this protocol show the near absorbance range between 0.1 and three. We compared several equations of spectrophotometric phycobiliproteins quantification known from the literature.
The equation of Bennett and Bogorad provided the best reconstruction of both phycocyanin and allophycocyanin content in the pigment standards with known protein concentrations. The differences between the individual equations are connected with variations in phycobiliproteins extinction coefficients of the specific organism. The use of streptomycin sulfate didn't lead to reduction of chlorophyll A in samples where sonication was applied or avoided.
As the representative data, we determined content of phycobiliproteins in Synechocystis cells. Phycobiliproteins content decreased under increasing light. The protocol was established to minimize time and equipment requirements for the written pigment analysis that can be performed in any standard live-science laboratory.
Also, the protocol contains two other steps, such as freeze drying of the cell pellet and proteins extraction. The total labor time for the phycobilisomes quantification is no longer than two hours.
