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Diana Miculescu
Introduction
Plants convert light energy into chemical energy by the process of photosynthesis. The
products of photosynthesis are then released to fuel the plant’s activities. During photosynthesis,
chloroplasts in the plant’s leaves capture light energy to generate nicotinamide adenine
dinucleotide phosphate and adenosine triphosphate that later aid in producing glucose by way of
the Calvin cycle. The Calvin cycle is made up of reduction/oxidation reactions which converts
carbon dioxide and other compounds into glucose for the plant to use.
One phase of the Calvin cycle is carbon fixation in which carbon dioxide is converted
Rubisco is found in the chloroplast of plants and is the carbon dioxide-fixing enzyme in the
Calvin cycle (Donnelly et al., 2014). Rubisco is the most abundant protein in the biological
world and has generated much interest because of its poor specificity and its slow catalytic rate
that limits the enzyme’s photosynthetic efficiency (Donnelly et al., 2014). This key role that
Rubisco plays in carbon fixation makes it a target for genetic manipulation (Andersson &
Backlund, 2008). In order to study the protein, Rubisco must first be extracted from the
chloroplasts and isolated from other proteins. Rubisco is highly soluble and is composed of a
large 55kDa subunit and a small 14kDa subunit (Salvucci et al., 1987). The objective is to extract
the proteins from spinach leaves employing the Ammonium Sulfate Precipitation method, isolate
Rubisco from the pellet using ion exchange column chromatography, and ensure Rubisco is
actually the protein isolated by using gel electrophoresis and spectrophotometry scan from 200 to
400 nm.
solubility. In this experiment, we will reach 37% and 50% saturation of ammonium sulfate. At
PROTEIN ISOLATION REPORT 3
high salt concentrations, proteins precipitate out of solution. The precipitate can then be
centrifuged and collected in pellet form. The pellet contains all the proteins that have
precipitated, and further techniques must be carried out to separate the proteins and isolate
Rubisco.
charge. The column used in this experiment is DEAE cellulose fast flow column with resin in the
column that contains beads covalently bound to positively charge N+H(C2H5)2 side chains. This
allows the negatively charged Rubisco protein to stick to the positively charged beads in the
column. Then, loading the low, medium, and high salt buffers, Rubisco should be released from
the beads because of the enzyme’s negative charge. To examine whether Rubisco has actually
been extracted, further techniques must be employed. Then, a spectrophotometry scan from 200
to 400 nm will be used to identify Rubisco by its spectra signature at 220 nm and 280 nm.
To ensure that enzyme isolated is in fact Rubisco, gel electrophoresis and SDS-PAGE
will be used because it allows one to analyze the purity of the sample in multimeric, dimer, or
monomer subunits of Rubisco. The hypothesis is the fractions with the highest absorbance will
form two detectable bands on the gel page corresponding to Rubisco’s two subunits: 55 kDa and
14kDa. In addition, the second pellet containing the high salt buffer should exhibit the most
Methods
About 300 g of fresh spinach leaves were de-ribbed and homogenized in 200 mL of
Buffer 1 (0.01 M K-PO4 buffer, pH 7.5, 0.3 mM EDTA, and 30 g/L polyvinylpolypyrrolidone)
PROTEIN ISOLATION REPORT 4
for one minute using a commercial blender on medium speed. The resulting suspension was
filtered through Miracloth. The 50 g of remaining filtrate was stirred for 15 min and saturated to
30% with solid ammonium sulfate, and the solution was centrifuged for 15 min at 9,000 x g at 4
°C. The supernatant was poured in a beaker and labeled Supernatant 1 (S37). The pellet was
suspended in 4 mL of distilled water and transferred into a dialysis bag. The pellet was dialyzed
against distilled water and labeled Pellet 1. The remaining filtrate in the beaker was stirred for 15
min while solid ammonium sulfate was added to reach 50% saturation. The filtrate was
centrifuged for 15 min at 9,000 x g at 4 °C. The supernatant was labeled Supernatant 2 (S50).
The pellet was dissolved in 4 mL of distilled water and dialyzed against distilled water, then was
Two positively charged DEAE Cellulose fast flow columns were obtained and
equilibrated with 30 mL Buffer A (10 mM Tris pH 8.0, 3 mM EDTA). The solid precipitate from
the dialyzed samples was centrifuged at 1000 rpm for 3 min. P1 was diluted 100X and 3-5 mL of
each sample (diluted P1 and undiluted P2) were loaded into separate columns and allowed to
flow through the column. The column was washed with 10 mL of Buffer A. After Buffer A ran
through, 10 mL of the low salt buffer (Buffer A + 50 mM NaCl) was loaded into each column.
Cuvettes collected about 2 mL fractions and numbered in order of collection. The scanning
spectrophotometer was blanked using the low salt buffer. The absorption at 280 nm for each
fraction was taken. The fraction with the highest optical density at 280 reading was transferred to
an Eppendorf tube. The procedure was repeated using a medium salt buffer (Buffer A + 200 mM
NaCl) and a high salt buffer (Buffer A + 500 mM NaCl). The column was washed with a resin
cleaning buffer.
PROTEIN ISOLATION REPORT 5
Results
The fractions of the low, medium, and high salt buffers from the ion exchange columns
were measured for highest absorbance. Of the low, medium, and high salt washes of Pellet 37%
saturation and the Pellet 50% saturation, high salt collectively contained the most protein (Table
1). Comparing the high salt washes for 37% and 50% saturation, the 50% high salt washes both
had higher absorbance values, showing that the 50% washed contained more protein than the
The absorbance values of the nine samples were taken with a spectrophotometry from
200 nm to 400 nm at 1-nm intervals (except Filtrate was taken at 10 nm intervals) to determine
protein’s spectra profile. The major absorbance for Filtrate occurred around 210-220 nm and
then the absorbance values declined after 280 nm (Figure 1A). For Supernatant 1, there was no
significant peak and the absorbance values increase and decrease until about 300 nm and then
decreased gradually (Figure 1B). For Pellet 2, absorbance values peaked after 280 nm, but the
values did not gradually decline after like the other samples (Figure 1C). The absorbance
spectrum for Pellet 1 with the High Salt wash increased and decreased until about 280 nm, then
PROTEIN ISOLATION REPORT 6
the absorbance decreased sharply (Figure 1D). For Pellet 2 washed with High Salt, there was a
significant peak after 280 nm and then decreased gradually (Figure 1E).
0.1
0.05 Abs
0
200 240 280 320 360 400
-0.05
Wavelength (nm)
1A) Filtrate: The fraction was measured using a spectrophotometer between 200 and 400 nm at 10-nm
intervals.
0.15
Absorbance
0.1
0.05
Abs
0
-0.05 200 240 280 320 360 400
Wavelength (nm)
1B) Supernatant 1: The fraction was measured using a spectrophotometer between 200 and 400 nm at 1-
nm intervals (graph shows every 10-nm).
0.8
0.6
0.4 Abs
0.2
0
200 240 280 320 360 400
Wavelength (nm)
1C) Pellet 2: The fraction was measured using a spectrophotometer between 200 and 400 nm at 1-nm
intervals (graph shows every 10-nm).
PROTEIN ISOLATION REPORT 7
0.2
Absorbance
0.1
0 Abs
200 240 280 320 360 400
-0.1
-0.2
Wavelength (nm)
1D) Pellet 1 High Salt: The fraction was measured using a spectrophotometer between 200 and
400 nm at 1-nm intervals.
0.6
0.4
Abs
0.2
0
200 240 280 320 360 400
Wavelength (nm)
1E) Pellet 2 High Salt: The fraction was measured using a spectrophotometer between 200 and
400 nm at 1-nm intervals (graph shows every 10-nm).
0.5
Abs
0
200 240 280 320 360 400
Wavelength (nm)
1F) Pellet 1: The fraction was measured using a spectrophotometer between 200 and 400 nm at
1-nm intervals (graph shows every 5-nm).
PROTEIN ISOLATION REPORT 8
Figure 1: Absorbance spectra of Pellet 1 and 2. The fraction was measured using a
spectrophotometer between 200 and 400 nm.
Filtrate (A), Supernatant 1 (B), Pellet 2 (C), Pellet 1 High Salt (D), Pellet 2 High Salt (E)
After UV spectrum analysis, the samples were loaded on an electrophoresis page and
analyzed for protein identification based on size. The four relatively bright bands on the ladder
lane are standards used as a comparison for the other bands (Figure 6). The four bands
correspond to the molecular weights of 14, 31, 66, and 97 kDa. The bands in the P2 High lane
contain the Pellet at 50% saturation with high salt wash. The lane showed two major bands and
one other band not as bright. The larger band was seen between the 66 and 31 kDa mark of the
ladder standard and the smaller one was seen near the 14 kDa mark. In addition, the bands in the
P2 lane were larger and brighter than the bands seen in the P2 High Lane. The number of bands
decreased from left to right from the lane labeled filtrate to High Salt Lane of Pellet 1. As with
Pellet 2, the bands also decreased from the Pellet 2 Lane to the Pellet 2 High wash Lane. The
bands in the Pellet 2 High wash Lane appeared more clear and defined than the bands in the
Pellet 2 and Pellet 2 Medium Salt wash Lanes. Using the line of best fit, the band the travelled
the farthest in the P2 High Lane was calculated to be 12.84 kDa (Figure 2A, Figure 3A).
PROTEIN ISOLATION REPORT 9
97
66
31
14
y = -3.6792x + 8.5281
4 R² = 0.9974
3
Distance Migrated
2
1 Linear (Distance
Migrated)
0
0 0.5 1 1.5 2 2.5
Log MW
𝑦 = −3.692𝑥 + 8.5281
−4.0781 = −3.6792𝑥
1.1084 = 𝑥
𝑥 = 𝐿𝑜𝑔 𝑀𝑊
1.1084 = 𝐿𝑜𝑔 𝑀𝑊
𝑀𝑊 = 12.84 𝑘𝐷𝑎
Figure 2. Gel Electrophoresis of Fractions, Standard Curve of Ladder, and Calculation of Band
Size
Discussion
The purpose of this experiment was to extract the protein Rubisco from spinach leaves
employing the Ammonium Sulfate Precipitation method, isolate Rubisco from the pellet using
ion exchange column chromatography, and ensure Rubisco was actually the protein isolated by
using gel electrophoresis and the spectrophotometry scans from 200 to 400 nm. It was predicted
that of the two salt-saturated solutions produced from the spinach extract (37% saturation and
50% saturation), Rubisco would precipitate in the second pellet, or the higher-concentrated
solution, due to its strong solubility. Referring to Table 1, the collective absorbance values for
the high salt washes of Pellet 50% saturation are greater than any other fractions, suggesting that
the protein concentration is greater in the in Pellet with 50% saturation. The results in Table 1
support the hypothesis that the most protein would be contained in the high salt washes or the
50% saturation fractions. In addition, the UV spectrum for Pellet 2 High Salt exhibits one
significant peak after 280 nm (Figure 1E). The absorbance spectrum for Pellet 2 High Salt should
have exhibited two peaks, one at 220 nm and another at 280 nm. A possible reason why two
PROTEIN ISOLATION REPORT 11
peaks were not observed could be because the spectrometer may not have scanned the samples
properly. This must be the case, since the bands characteristic to Rubisco are seen in the Pellet 2
To further support the initial hypothesis of expecting Rubisco subunits to be found in the
High Salt wash of Pellet 2, the SDS-PAGE illustrates the most distinct bands in the P2 High lane
(Figure 2A), which means that most of the Rubisco eluted from the spinach extract in the 50%
concentrated solution. This supports the hypothesis that Rubisco will elute from the spinach
extract in the 50% solution as opposed to the 37% solution. The observation that no distinct
bands can be seen in in the Pellet 1 lanes of Figure 2A supports that Rubisco eluted mostly in the
50% saturated solution rather than the 37% saturation solution. The gel page shows two distinct
bands in the P2 High lane which correspond to the molecular weights of the subunits of Rubisco:
14 kDa and 55 kDa. The band that migrated furthest in the Pellet 2 High Salt lane was calculated
to have a molecular weight of 12.84 kDa (Figure 2C). This molecular weight compares to the 14
kDa subunit of Rubisco. The slight difference in molecular weight may be caused by inaccurate
measurement of the distance migrated since the UV image shows a curved edge on the right side
(Figure 2B). This slight curve caused the migration of the band to seem longer, and therefore
Overall, the experiments demonstrated that Rubisco could be isolated using the proper
chromatography and verifying that Rubisco was truly extracted by using SDS-PAGE and
spectrophotometry. Furthermore, since the experiment was successful in extracting Rubisco, the
basis of each technique used, such as solubility and charge are promising in future experiments
to extract other proteins. Successful extraction for proteins is significant in the scientific world
PROTEIN ISOLATION REPORT 12
because these proteins can now be studied, manipulated, and experimented with to gain a greater
References
Andersson, I. & Backlund, A. (2008). Structure and function of Rubisco. Plant Physiology and
Biochemistry, 46:3:275-291.
Donnelly, K.O., Zhao, G., Patel, P., Butt, M.S., Mak, L.H., Kretchmer, S., Woscholski, R., Bart,
Salvucci, M.E., Werneke, J.M., Ogren, W.L., Portis, A.R. (1987). Purification and Species
Spreitzer, R.J. & Salvucci, M.E. (2002). Rubisco: Structure, Regulatory Interactions, and