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Ribulose-1,5-bisphosphate carboxylase oxygenase, known as Rubisco, is a protein most prevalent in plants because of its enzymatic capabilities for sugar anabolism in the dark reactions of photosynthesis (Goodsell 2000). The enzyme is composed of two subunits with molecular weights of 55 kDa and 13 kDa and a predominantly negatively charged surface area (GSU Lab Manual 2010). Scientific techniques were utilized for proper isolation and purification of Rubisco from spinach leaves.
In order to isolate proteins from the spinach leaves, ammonium sulfate salts were used to precipitate these proteins by reducing their reactions with water. Increasing the salt concentration will decrease protein solubility because the water molecules will interact with ammonium sulfate. As a result, protein molecules start to aggregate together and form a desired precipitate that was placed in a centrifuge to obtain a pellet. Locating the state of matter of Rubisco at different saturation levels reveals the protein's solubility.
The next technique was ion exchange column chromatography, which further isolated Rubisco from other proteins in the pellet by ionic charges. Using columns coated in positively charged beads trapped negatively charged proteins, of which are expected to be Rubisco. The results further characterize the degree of negative charge of the protein.
Then, an SDS-PAGE Gel Electrophoresis was conducted to identify the sizes of proteins present after ion exchange. On the spectrum, small proteins migrate farther and faster from the point of injection, while large proteins move slower and stay close in proximity to the wells. Application of SDS guarantees that the proteins all have the same degree of negative charge, so the gel displays only information about the protein's molecular weight after thorough analysis. Graphing the distances the marker bands traveled by the logarithm of the molecular weight, results in a best fit line, which serves as a standard curve. Plotting the actually molecular weight would create an exponential curve; so calculating the logarithm of the sizes will result in points that produce a best fit line. Utilizing the graph, the approximate molecular weights of the proteins present in the gel will be shown. These techniques will further confirm that Rubisco has been successfully isolated, by characterizing its solubility, charge, and molecular weight. A spectral analysis was the final step in the determination of proper Rubisco isolation. Since Rubisco has a high solubility, with a predominantly negatively-charged surface area, then gel electrophoresis should convey that Rubisco resides heavily in the P2 high sample, with bands around 55 kDa.
A weight of 300 grams of spinach were measured, de-ribbed, and homogenized by using a blender in 200 mL of Buffer 1, consisting of 0.01 M KPO4 buffer, 0.3 mM EDTA, and 30 grams/L of polyvinylpolypyrrolidone. Using Miracloth, a pocket was formed over a beaker, and the suspension was filtered with the aid of a pipette bulb to obtain most of the filtrate. Then, 6.3 grams of ammonium sulfate, which was calculated to reach 37% saturation, given 210 g/L of solution, was slowly added to the filtrate while it was stirring for duration of 15 minutes. Next, it was placed in a centrifuge for 30 minutes at 9,000 g to bring down proteins to form a pellet. The supernatant was carefully poured into cylinder, where its estimated volume was measured and transferred to a beaker. The amount of ammonium sulfate, of 2.975 grams, required to reach 50% saturation was calculated, given 85 g/L of solution and added slowly to the supernatant, while it was stirring for 15 minutes. The pellet was resuspended in 4 mL of water and transferred into a dialysis bag, labeled P1, to dialyze against water for rehydration. The supernatant was sent to the centrifuge for 30 minutes at 9,000 g to bring the proteins from the precipitate down towards the bottom of the tube. The supernatant was discarded, and the pellet, labeled P 2, was dissolved in
4 mL of water and placed in a dialysis bag.
During ion exchange column chromatography, two columns were equilibrated with
30 mL Buffer A. While the columns were equilibrating, 1 mL each of P1 and P2 were transferred to labeled Eppendorf tubes, which served as controls. Then, 5 mL of both P1, which was diluted
100-fold, and P2 were loaded into each of the two columns. Each column was washed with 10 mL Buffer A. After allowing Buffer A to flow, 10 mL of low salt buffer was poured into both columns. Fractions were collected from the columns into cuvettes (three-fourths full) that have been labeled and numbered. Each of the cuvettes underwent the spectrophotometer, and the OD280 readings were obtained. After, 1.5 mL of the fraction samples of P1 and P2 with the highest OD280 were obtained and transferred to labeled Eppendorf tubes. A second round of
10 mL of medium salt elution was poured into both columns, and fractions were collected in the same manner. The OD280 levels of those fractions were obtained, and of those with the highest OD280, 1.5 mL from P1 and P2 was drawn out and transferred to labeled Eppendorf tubes. A final high salt concentration of chlorine ions was poured into both columns. Fractions, OD280 readings, and 1.5 mL samples containing the highest OD280 were collected in the same manner.
To prepare for SDS-PAGE Gel Electrophoresis, 60 Î¼L of each of the 9 samples (marker, P1, P1 low, P1 medium, P1 high, P2, P2 low, P2 medium, P2 high) were transferred to separate Eppendorf tubes. Then to each tube, 30 Î¼L of dye was added. All samples were heated at 95Â°C for 5 minutes. The wells of the gel were filled with 10 Î¼L of the marker and 25 Î¼L of the remaining samples. The gels ran at 180V for 50 minutes. Then, the gel was carefully removed from the glass plate sandwich and set in Coomassie Blue, a stain-fixing protein, for 30 minutes. Next, the gel was placed in Destain overnight. After the gels had dried, each of the bands was measured by the distance they traveled from the wells. The distance the bands of the marker
traveled were measured and plotted against the logarithm of molecular weights (97 kDa, 66 kDa,
45 kDa) to produce a standard curve. To determine the approximate weights in Daltons of the proteins in the pellets, the inverse logarithm was taken from the x-value of the standard curve.
The gel showed a protein present in P1 high just under the 45 kDa mark, which was calculated to be around 42 kDa, using the standard curve. The band revealed that the protein has a low solubility because the protein had precipitated at 37% saturation. Given that the protein was found in the pellet where the high salt buffer took place, this indicated that the protein is highly negatively charged, since it took a high salt concentration to elude the protein.
In the P2 column, a heavy set of bands appeared to be merging together, so an individual band was not measured. The P2 low column showed a light, yet distinctive band, whose molecular weight was calculated as 36 kDa. The intensity of the band illustrated that there resided small amounts of this protein. The location of the band characterized the protein to be highly soluble because it precipitated in a solution that was 50% saturated. Since this protein was found after a low salt elution, it has weak negative charge because a low salt concentration of chlorine ions was enough to wash away this protein. The P2 medium band was slightly darker in intensity than P2 low, indicating a larger amount of protein present than in P2 low. The band appeared farther in distance than P2 low, calculating at a molecular weight of 38 kDa. The protein has a high solubility because it appeared in a pellet at 50% saturation. It has a moderate negative charge after going through a medium salt concentration buffer. Under the P2 high column, there appeared to be a highly dense band between 66 kDa and 45 kDa, where Rubisco is expected. According to the standard curve, the band's approximate molecular weight was calculated as roughly 50 kDa, roughly the same size as Rubisco. The intensity implies that the protein was abundant in the sample, with a high solubility because the protein precipitated at a high saturation level of 50%. Since the protein was found present after a high salt elution, this describes the protein's strong negative charge because the protein was isolated after a high salt concentration. Figure 6 shows a strong peak at wavelength 230 nm, implying that the protein present is best measured at this wavelength. Absorbance at this particular wavelength of 230 nm indicates that Rubisco is definitely present in the P2 high sample (Liang et al. 2007). Overall, Rubisco was successfully isolated from spinach leaves.
Figure 1 shows the measurements (in millimeters) of the distances each band has traveled along the gel. P2 high contained an immensely thick band that traveled 22 mm from the well. P2 medium contained a moderate band, which traveled 28 mm, while P2 low had a low intensity band that traveled 26 mm. P2 band traveled 29 mm, whereas P1 high traveled 26 mm. There were no visible bands in P1 medium, P1 low, and P1. The far most left lane contained 3 light bands that were considered initially as the marker, but later concluded those bands to be overflow of the standard marker. Proteins were most prevalent in P2 solutions
Figure 2 provides the approximate molecular weights of the bands when taking the inverse logarithm of the pinpointed x-value. P1 high and P2 low were both 42 kDa. P2 had a molecular weight of 36 kDa, P2 medium had a weight of 38 kDa, and P2 high had a weight of
50 kDa. The graph has a negative slope, depicting that distance and molecular weight are indirectly proportional. A decrease in distance traveled indicates larger size of the protein.
Figure 3 displays that P1 low obtained zero absorbance within the range of
200 nm-320 nm, which indicated that no protein exists in this sample.
Figure 4 shows that P1 high obtained zero absorbance as well within the 200 nm-320 nm range, which indicated that no protein exists in this sample.
Figure 5 also shows that P2 low obtained zero absorbance within the range of 200 nm-300 nm, which indicated that no protein exists in this sample.
Figure 6 shows an optimum peak with an OD reading of 2.52 at 230 nm, then to a sudden drop to zero absorbance around 260 nm.
Figure 1: SDS-PAGE Gel Electrophoresis of Pellet 1 & Pellet 2. First, 60 Î¼L of the 9 samples were measured, and 30 Î¼L of dye was added. All samples were heated at 95Â°C for 5 minutes. Then, 10 Î¼L of the standard marker was added into the first well. Next, 25 Î¼L of each of the remaining 8 samples (P2 high, P2 medium, P2 low, P2, P1 high, P1 medium, P1 low, and P1) were injected into the gel, which ran under 180V for 50 minutes.
Figure 2: Negative Correlation Between the Distance of Bands Traveled and Logarithm of Molecular Weight. The distances traveled were measured in millimeters from the well to the middle of each individual band. The given molecular weights of the marker was 97 kDa, 66 kDa, and 45 kDa. The logarithm of these weights were calculated and plotted. A best fit line was drawn in order to find the approximate weights of P1 high, P2, P2 low, P2 medium, and P2 high.
Figure 3: A spectral analysis of P1 low reveals no protein present in sample.
Figure 4: A spectral analysis of P1 high reveals no protein present in sample.
Figure 5: A spectral analysis of P2 low reveals no protein present in sample.
Figure 6: A spectral analysis of P2 high reveals a protein is present and is best measured at wavelength 230 nm.
Goodsell, David S. 2000. Rubisco.Feb 23 2010. < http://www.rcsb.org/pdb/static.do?
GSU Lab Manual. Georgia State University. 2010.
Liang, Xiao, et al. Effect of Mg2+ on the Structure and Function of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase. Humana Press Inc: 2007.