Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis

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 temperature for 5 min), the supernatant was discarded, and the samples were either stored at -80 °C and freeze-dried as preparation for the individual steps of this protocol or stored at -20 °C or -80 °C for comparative extraction and quantifications analysis.

The tested extraction procedures included cell disruption by sonication, homogenization with zirconia beads within the PBS buffer, and freeze-thawing. The results of different extraction methods are summarized in Figure 1. The method as described within this protocol provided the highest phycobiliproteins yields together with the lowest chlorophyll a contamination in the extraction buffer. Chlorophyll a was detected in the extraction buffer when sonication was used for the cell disruption. Repeating cycles of freezing and thawing the samples in an ultrasound bath on ice resulted in higher protein yields in the extraction buffer. However, at the same time, the concentration of chlorophyll a in the extraction buffer increased (Figure 1 inset), which excluded a proper phycobiliprotein content quantification.

The optimal homogenization time was 15 s. Homogenization for both shorter (5 s) and longer (20 s) periods provided slightly lower phycobiliprotein yields, as shown in Figure 2. Homogenizing the cells with glass beads of a diameter of 2 mm resulted in the highest yields when compared with glass beads of a diameter of 0.5 mm, 1 mm, or 4 mm, with zirconia beads of a 0.5-mm diameter, or with sea sand grains. The PBS extraction buffer was tested as the optimal buffer for phycobiliproteins extraction (Figure 3). The addition of 100 mM NaCl or 150 mM KCl either to the PBS buffer or to water did not increase phycobiliprotein extraction efficiency, and the same went for using 20 mM sodium acetate buffer for extraction (Figure 3). The optimal phycobiliprotein extraction time was 60 minutes because, after more time (up to 240 minutes), the phycobiliprotein concentration in the extraction buffer did not change significantly (Figure 4).

We also tested the spectrophotometer linearity range for the phycobiliprotein measurement (Figure 5) and compared several equations of phycobiliprotein quantification, using both phycocyanin and allophycocyanin standards (Figure 6). The spectrophotometer used in this protocol showed a linear absorbance range between 0.1 – 3.0 (Figure 5). Since the spectra of the protein extract had its maximal absorption around 620 nm (A₆₂₀), and at 652 nm, the absorption (A₆₅₂) was only 33% of A₆₂₀ (Figure 7), the spectrophotometer linearity for A₆₅₂ (maximum allophycocyanin absorbance) was tested only up to A₆₅₂ of 1.16. The equations (1) and (2) of Bennett and Bogorad¹² provided the best reconstruction of both phycocyanin and allophycocyanin standards among all tested equations (Figure 6). Streptomycin sulfate, used in several previous studies for the elimination of membrane fragments containing chlorophyll a, was shown as not necessary for the extraction (Figure 7).

For the representative experiment, Synechocystis was cultivated in a photobioreactor²⁵ in a turbidostat regime, where the cells were maintained in exponential growth phase within a defined range of optical density (measured at 680 nm, OD₆₈₀) by a controlled dilution by a fresh cultivation medium (BG11 medium buffered with 17 mM HEPES)^11,26. The cultures were cultivated at 32 °C and the input air contained 0.5% CO₂. The cultures were illuminated with a red light (λ_max: 633 nm, λ_1/2: 20 nm) of an intensity of 25 – 1100 µmol(photons)/(m²·s), together with a blue light (λ_max: 445 nm, λ_1/2: 20 nm) of an intensity of 25 µmol(photons)/(m²·s). The optical density of the culture suspension was measured by the photobioreactor base, and the range of OD₆₈₀ was set to 0.52 – 0.58 (2 x 10⁷ to 4 x 10⁷ cells/mL). The cultures were cultivated under each specific light intensity for at least 24 hours to provide enough time for acclimation. After reaching growth stability, the cultures were sampled for dry weight (according to steps 2.1 – 2.8 of this protocol), cell count, and phycocyanin and allophycocyanin content. The results of the phycobiliprotein evaluation under increasing light intensities are shown in Figure 8. Synechocystis decreased the phycobiliprotein content from 12% of cellular dry weight (505 fg/cell) under the lowest irradiance to 3% of cellular dry weight (280 fg/cell) under the highest irradiance.

Figure 1: Comparison of the extraction efficiency of the method described in this protocol with several reference methods. The concentration of phycocyanin (black bars) and allophycocyanin (white bars) in the crude protein extract was measured after extraction in the PBS buffer for 60 min. Method A is described within this protocol. Method B was modified as follows: after harvesting, the cells were centrifuged (15,000 x g at laboratory temperature for 5 min) and stored at -20 °C overnight. The frozen samples were sonicated for 120 s at 4 °C, and the phycobiliproteins were extracted in PBS buffer for 60 min. Method C was modified from method B by storing the pellets of harvested cells at -80 °C for 30 min, freeze-drying the cellular pellets, and adding PBS buffer to the dry pellets. Method D was modified from Chung et al.¹⁶: after freeze-drying (same as in method C), 0.25 mL of Na-acetate with 1% streptomycin sulfate buffer (pH 5.5) was added to the dry cellular pellet, together with 0.25 mL of zirconium beads (with a diameter of 0.5 mm), and the samples were homogenized twice for 60 s on the homogenizer. After homogenization, another 0.5 mL of Na-acetate with 1% streptomycin sulfate buffer (pH 5.5) was added to the sample, the tubes were vortexed, and the proteins were extracted on ice for 30 min. Method E consisted of a single freezing-thawing cycle in PBS buffer and protein extraction for 24 h at 4 °C. Method F (inset figure) consisted of one and six cycles of freezing the cells (initially, freeze-dried and resuspended in PBS buffer) in liquid nitrogen, followed by sonication on ice. Due to significant chlorophyll a presence in the crude protein extract, the phycobiliproteins were not quantified within method F. The depicted values represent averages from three technical replicates, with the error bars representing standard deviations. Pigment concentrations were calculated according to equations (1) and (2) of this protocol. Please click here to view a larger version of this figure.

Figure 2: Effect of the homogenization time on the phycobiliprotein extraction efficiency. The lyophilized pellets of Synechocystis cells (according to steps 3.1 – 3.5 of this protocol) were disrupted with glass beads on homogenizer for 5 s, 15 s, and 20 s. The depicted values represent averages from three technical replicates, with the error bars representing standard deviations. Please click here to view a larger version of this figure.

Figure 3: Phycobiliprotein extraction efficiency in various extraction buffers. The phycobiliproteins were extracted in the PBS buffer, in the PBS buffer with an addition of 100 mM NaCl or 150 mM KCl, in deionized water with 100 mM NaCl or 150 mM KCl, according to Hemlata and Fareha²², and in 20 mM sodium acetate according to Chung et al.¹⁶. The extraction was performed at 4 °C according to steps 4.1 – 4.3 of this protocol. The depicted values represent averages from three technical replicates, with the error bars representing standard deviations. Please click here to view a larger version of this figure.

Figure 4: Phycobiliprotein stability in the extraction buffer in time. After the extraction procedure, performed as described in steps 4.1 – 4.3 of this protocol, the phycobiliprotein extract in PBS buffer (pH 7.4) was stored at 4 °C for up to 4 hours, with no significant phycocyanin (circles) and allophycocyanin (squares) degradation. The dashed line represents the linear fit of the time points calculated by the least squares method. The depicted values represent averages from three technical replicates, with the error bars representing standard deviations. Please click here to view a larger version of this figure.

Figure 5: Representative measurements of phycocyanin and allophycocyanin concentrations in Synechocystis. A dense culture suspension (phycocyanin: 456 µg/mL, allophycocyanin: 106 µg/mL) was gradually diluted up to a concentration of 19 µg/mL for both phycocyanin and allophycocyanin. The depicted values represent averages from three technical replicates, with the error bars representing standard deviations. The dotted line represents the linear fit of the concentration points calculated by the least squares method. Please click here to view a larger version of this figure.

Figure 6: Estimation of phycocyanin and allophycocyanin concentrations in pigments standards. The content of phycocyanin and allophycocyanin in pigment standards was measured spectrophotometrically according to the equations of Bennett and Bogorad¹², Lüder et al.¹³, Sampath-Wiley and Neefus¹⁵, Evans¹⁴, and Chung et al.¹⁶. Protein content in the standards (as necessary for phycobiliproteins determination) was quantified using a solution of bicinchoninic acid, sodium carbonate, sodium tartrate, and sodium bicarbonate in 0.1 N NaOH (with a final pH of 11.25), in reaction with 4% (w/v) copper(II) sulfate pentahydrate²⁷, using bovine serum albumin as a protein standard. The equations of Bennett and Bogorad provided the highest reconstruction of both pigments: 96% of phycocyanin standard and 99% of allophycocyanin standard. The depicted values represent averages from three technical replicates, with the error bars representing standard deviations. The dashed line highlights the 100% point. Please click here to view a larger version of this figure.

Figure 7: The effect of streptomycin sulfate on the absorption spectrum of the crude protein extract in the PBS buffer. (A) The extraction procedure as described in steps 4.1 – 4.3 of this protocol or (B) using sonication as described in the legend of Figure 1 for Method F was performed in PBS buffer (circles) and in PBS buffer supplemented with 1% streptomycin sulfate (squares). The phycobiliprotein quantification was performed according to steps 5.1 – 5.4 of this protocol. The depicted values represent averages from three technical replicates, with the error bars representing standard deviations. Please click here to view a larger version of this figure.

Figure 8: Concentration of phycocyanin (circles) and allophycocyanin (squares) in Synechocystis cultivated under red-orange light of an intensity of 25 – 1100 µmol(photons)/(m2·s). Synechocystis cultures were cultivated in a photobioreactor at 32 °C, with 0.5% CO₂ in the input air, in BG11 cultivation medium supplemented with 17 mM HEPES in a turbidostat regime according to Zavrel et al.¹¹. The phycobiliprotein concentrations were measured according to this protocol and the final phycobiliprotein content was normalized per cellular dry weight, as described in section 2 of this protocol. The dashed lines represent the power fit of the measured points, calculated by the least squares method. The depicted values represent averages from three technical replicates, with the error bars representing standard deviations. Please click here to view a larger version of this figure.