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Christian Walleck
Partner: Avery Brothers 12/5/13 Section 2 Isolation and Purification of Pseudanabaena Phycobiliproteins

Introduction
The goal of this experiment is to isolate and partially purify the phycobiliproteins phycocyanin, allophycocyanin, and phycoerythrin from photosystem II of the cyanobacteria Pseudanabaena 7409. The starting sample is a mixture of proteins obtained from lysing Pseudanabaena cells and subsequently precipitating the protein with ammonia sulfate and concentrating with dialysis. These steps were completed before the experiment began. Phycobiliproteins were first separated by anion-exchange column chromatography. Net negative charge proteins stick to the column while net positive proteins flow through. Bound proteins were then eluted by salt fractionation. Proteins with smaller net negative charge were expected to elute faster than higher net negative charge proteins. About ten collected fractions of the same color, blue or pink, were pooled and saved for additional purification. The samples were separated further by centrifugation. Multiple rounds of centrifugation removed proteins with too high or too low molecular weights. Remaining sample was placed in a 7000MWCO dialysis unit overnight allowing molecules under 7000Da to diffuse out. After dialysis, the samples were ready to be tested by SDS-PAGE. The second purpose of this experiment was to prove that the desired phycobiliproteins were the proteins obtained through purification. The first test was spectrophotometry of the pink and blue samples obtained from column chromatography. An absorbance spectrum from 400nm to 750nm was obtained for comparison to the known spectra of the phycobiliproteins. SDS-PAGE was the other method used to determine the composition the two protein samples. The samples were run on a gel against rainbow marker and samples of pure allophycocyanin, phycocyanin, and phycoerythrin. Bands on the gel were observed under ultraviolet light and stained with coomassie brilliant blue dye. The identity of each isolated protein was determined using the SDSPAGE gel and absorbance spectra.

Methods and Materials

Pseudanabaena 7409 protein sample was used. Proteins were already separated from other cell components before the experiment.

Ion Exchange Chromatography

The sample was separated using anion-exchange column chromatography first. The column was packed with DEAE sephacel resin. The column was washed with 30ml of isolation buffer (25mM tris, 5mM EDTA, pH 7) before addition of sample. Sample was added and washed with an additional 30ml isolation buffer until pink color began to elute. At this point, a pump, gradient mixer, and fraction collector were set up. The gradient mixer contained 100ml NaCl limiting buffer (350mM NaCl) in the outer chamber and 100ml isolation buffer in the inner chamber. Flow rate was set at 1.5ml/min and fractions were collected every 2 minutes. Eight fractions were pooled for both pink and blue solution with 1ml of each sample was set aside for spectrophotometry.

Phycobiliprotein Partial Purification

8.636g ammonia sulfate was added to the both samples, and they were centrifuged for 20 minutes at 7700g. Supernatant was removed and the pellets resuspended in 1ml ammonia sulfate solution (65% saturated). Then, samples were spun in a microfuge for 5 minutes at maximum speed. Supernatant was discarded and the pellets were resuspended in 100ul isolation buffer (25mM tris, 5mM EDTA, pH7). Samples were again spun for 5 minutes at maximum speed in the microfuge. The supernatant was saved this time resulting in a blue sample and pink sample each with about 0.3ml of volume. The samples were transferred to dialysis units (MWCO 7000Da) and allowed to sit in isolation buffer overnight.

SDS-Polyacrylamide Gel Electrophoresis

The gel for SDS-PAGE was already made. The stacking gel consists of 3.9% polyacrylamide, produced by mixing 3.35ml water, 2.5ml Tris(1.5M, pH 8.8), 100ul SDS(10%), 4ml polyacrylamide (30%),

4 53ul ammonium persulfate (10%), and 6ul TEMED. The separating gel contains 12% polyacrylamide, produced by mixing 6.1ml water, 2.5ml Tris(.5M, pH 6.8), 100ul SDS(10%), 1.3ml polyacrylamide(30%), 53ul ammonium persulfate(10%), and 11ul TEMED. Samples were prepared by mixing 20ul of sample with 20ul reducing sample buffer (4ml water, 1ml Tris-HCl (.5M, pH 6.8), .8ml glycerol, 1.6ml SDS(10%), .4ml 2-B-mercaptoethanol, .2ml bromophenol blue(0.1%)). Then, protein samples were heated at 95C for 5 minutes. 20ul of allophycocyanin, phycocyanin, and phycoerythrin were placed in the gel along with 10ul of rainbow marker, pink sample, and blue sample. The gel was run in 1x electrophoresis running buffer (0.2M Tris buffer, pH 8.3, 0.2M glycine, 0.1% SDS) at 200V for about 45 minutes. The gel was placed in fixative solution (530ml water, 70 ml glacial acetic acid, 400ml methanol) for 5 minutes after SDS-PAGE. The gel was observed under ultraviolet light and then stained with additional fixative solution containing 0.05% coomassie brilliant blue dye.

Results
Salt fractionation resulted in a pink band and blue band eluting from the column. The pink band eluted very quickly, within the first 12 fractions. The blue band eluted later when the salt concentration increased. Eight fractions of each color were pooled together and used in the rest of the experiment. Table 1 shows which fractions were used. Table 1 Column Chromatography Fractions Fractions Pooled 2-9 18-25

Sample 1 2

Color Observed Pink Blue

A small amount of the pooled samples was removed to measure the absorbance spectrum from 400nm to 750nm. The pink sample had a maximum absorbance at 567nm and a very small peak at 543nm. The maximum absorbance matches closely with the expected maximum absorbance of

5 phycoerythrin (565nm) indicating phycoerythrin is the major component of the pink sample. The absorbance spectrum can be seen in figure 1. Figure 1

The blue sample had an absorbance maximum at 618nm, near the 615nm expected absorbance maximum for phycocyanin. The absorbance spectrum for blue sample is shown in figure 2 on the next page.

Figure 2

After more purification from centrifugation and dialysis, the samples were tested by SDS-PAGE. Unknown samples were tested along with phycobiliprotein standards and the molecular weight ladder rainbow marker. The gel was observed under ultraviolet light where the phycoerythrin standard and pink sample were florescent further indicating the unknown pink sample was phycoerythrin. Then, the gel was stained so all bands could be seen. The picture of the stained gel is shown in figure 3.

7 Figure 3 Lane 1 2 3 4 5 6 7 8 9 10
Lane 1- Empty Lane 2- Spirulina Allophycocyanin (20ul) Lane 3- Spirulina Phycocyanin (20ul) Lane 4- Red Algae Phycoerythrin (20ul) Lane 5- Rainbow Marker (10ul) Lane 6- Pink Sample (20ul) Lane 7- Blue Sample (20ul) Lanes 8-10 - Empty

The molecular weight of each band in the rainbow ladder and the distance they migrated was used to create a standard curve buy plotting log molecular weight against distance travelled. The graph is shown in figure 4. Figure 4

Molecular Weight Standard Curve

y = -0.026x + 1.6498 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0 10 20 Distance Travelled (mm) 30 40

R2=0.8802

Log MW

Series1 Linear (Series1)

8 The molecular weight of each band on the gel was then found using the standard curve. Below is a sample calculation of the first band showing how the molecular weight was calculated from the graph.

The experimentally determined molecular weights are shown in table3. Table 3 Distance Travelled and Molecular Weights Distance (mm) 20.5 22.5 17 20 30 16 18 25 18 21

Sample Allophycocyanin Allophycocyanin Phycocyanin Phycocyanin Phycocyanin unknown Phycoerythrin Phycoerythin Phycoerythrin Phycocyanin Phycocyanin

Molecular Weight (kDa) 13.1 11.6 16.1 13.5 7.4 17.1 15.2 10.0 15.2 12.7

After the molecular weight of each subunit was determined experimentally, the molecular weight of standard complexes containing multiple subunits could be found by adding up the weight of each subunit. Experimental complex molecular weights and their theoretical values are shown in table 4. Table 4 Complex Experimental and Theoretical Molecular Weights Subunit assembly Experimental MW(kDa) Theoretical MW(kDa) ()6 193.8 240 ()6 167.4 220 ()3 74.1 100

Protein R-Phycoerythrin C-Phycocyanin Allophycocyanin

The experimental weight of each complex was significantly lower than the theoretical value. Consistently low experimental values are likely due to poor separation on the gel, leading to inaccurate

9 data. The R2 value is 0.8802, meaning the linear fit is poor and molecular weights obtained from the data are inaccurate.

Discussion The original protein sample was successfully run through column and separated into two samples by color. After fractions were pooled, 65% saturated ammonium sulfate was added to the samples to force protein to precipitate out of solution before it was centrifuged. The ammonium sulfate occupies water molecules causing protein to aggregate and precipitate. Ammonium sulfate was the salt chosen because it is has a high solubility and it works over a range of pH. The samples were then purified through centrifugation and dialysis. Dialysis purified the samples by allowing small molecules below 7000Da to exit through a membrane leaving behind only larger molecules. However, it also diluted the sample because more buffer entered the dialysis tube. There was little difference in volume for the pink sample but the blue sample volume clearly increased from about .3ml to .4ml during dialysis. The column was packed with DEAE sephacel. It is used in anion-exchange due to its positive charge. Net negative charged proteins bind to the DEAE sephacel resin and move through the column more slowly than positively charged proteins. The elution buffer used was isolation buffer mixed in a gradient mixer with NaCl. Increasing salt concentration interfered with protein binding to the column and allowed it to elute. The most negative proteins would still elute last because it would take higher salt concentration to disrupt their binding to the column.

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The spectrum profile of the pink fraction indicates that phycoerythrin is present because of the matching absorbance maximums, which agrees with the SDS-PAGE data. The pink sample did not migrate the same distance as the phycoerythrin standard, but it was higly florescent indicating the presence of phycoerthrin. The blue sample had an absorbance maximum of 618 corresponding to phycocyanin. The gel similarly showed phycocyanin was the primary protein present; its two bands matched the two bands for phycocyanin standard very closely. Phycoerythrin is pink in solution. It eluted first from the column with a bright pink color as expected. Allophycocyanin is a light blue and phycocyanin is slightly darker blue in solution. The second color to elute from the column was light blue as expected. Lanes 4 and 6 fluoresced under ultraviolet light. Lane 4 contained phycoerythrin and lane 6 contained pink sample which was determined to contain phycoerythrin as well. The relative charge of each phycobiliprotein can be determined from their behavior in the column. Since phycoerythrin eluted first with little or no salt present, it has the most positive charge. Allophycocyanin and phycocyanin have a net negative charge because they required salt to elute from the column. Based on the gel, allophycocyanin is made of two the smallest subunits. Phycocyanin is made up of two slightly larger subunits; however, there is a third band present indicating the presence of a very small subunit under 10kDa. Phycoerythrin is made up of the two largest subunits with similar size. They barely separated on the gel. Iron binding protein remains attached to the column even after 300mM NaCl is washed over it because it has an extremely high affinity for positive charge. It is designed to bind iron which is a positive molecule. Since it binds so strongly to positively charged DEAE sephacel

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resin, salting out will not work. Changing the pH would be a better technique to elute iron binding protein because it would lose its strong attraction to the DEAE sephacel. There was one unexpected band on the SDS-PAGE gel. It was the band labeled unknown in the Spirulina Phycocyanin standard (Lane 3). It was the smallest protein on the gel, with an experimental molecular weight of 7.4 kDa. It is likely too small to be either subunit or of phycocyanin. The molecule is likely negatively charged because it is part of phycocyanin, and phycocyanin was the last protein to elute from the column.