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PROTEIN ISOLATION REPORT 1

Protein Isolation Final Report

Diana Miculescu

Georgia State University


PROTEIN ISOLATION REPORT 2

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

into usable carbon. Ribulose-1,5-biphsphate carboxylase/oxygenase, commonly known as

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.

Ammonium Sulfate Precipitation is a method used to purify proteins by basis of

solubility. In this experiment, we will reach 37% and 50% saturation of ammonium sulfate. At
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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.

Ion-Exchange Column Chromatography is a protein purification method by basis of ion

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

visible bands due to the enzyme’s highly negative charge.

Methods

Isolation by Ammonium Sulfate Precipitation

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)
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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

labeled Pellet 2 (P50).

Isolation by Ion Exchange Column Chromatography

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.
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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

37% saturation (Table 1).

Table 1. Optical Densities280 for Pellet 1 and Pellet 2 Fractions


To determine protein concentration of each sample was measured in a spectrophotometer at 280
nm, the optimal wavelength for detecting proteins. The greater absorbance value, the greater the
protein concentration in the fraction. *Highest absorbance readings
Pellet 37% Saturation (Pellet 1) Pellet 50% Saturation (Pellet 2)
Fraction Low Salt Medium High Salt Low Salt Medium High Salt
Salt Salt
1 0.012 0.011 0.108 0.173 0.130 0.380
2 0.103 0.004 0.123* 0.104 0.216 0.586
3 0.009 0.014 0.110 0.249* 0.313* 0.587
4 0.120* 0.050* 0.112 0.071 0.286 0.415*
5 0.024 0.029 0.094 0.213 -0.014 0.307

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
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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).

Absorbance vs. Wavelength for Filtrate


0.2
0.15
Absorbance

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.

Absorbance vs. Wavelength for Supernatant 1

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).

Absorbance vs. Wavelength for Pellet 2


1.2
1
Absorbance

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).
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Absorbance vs. Wavelength for Pellet 1 High Salt


0.3

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.

Absorbance vs. Wavelength for Pellet 2 High Salt


1
0.8
Absorbance

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).

Absorbance vs. Wavelength for Pellet 1


1.5
Absorbance

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).
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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).
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97
66
31
14

2A) Gel Electrophoresis


A standard ladder was loaded into the first lane to provide the bands for the subunits. The remaining nine
fractions were administered into the 10% acrylamide gel page in the order indicated below. The
electrophoresis was powered at 110 V for 50 minutes to generate an image.
Supernatant 1 (S1), Pellet 1 (P1), Pellet 1 Medium Salt (P1 Med), Pellet 1 High Salt (P1 High),
Supernatant 2 (S2), Pellet 2 (P2), Pellet 2 Medium Salt (P2 Med), Pellet 2 High Salt (P2 High)

Distance Migrated vs. Log MW


5
Distance Migrated cm

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

2B) Distance of Band Migration and Calculation of Molecular Weight


The distance of the bands labeled ladder seen in the gel electrophoresis image were measured in
centimeters starting from the top of the lanes. The distance of the bands migrated were plotted against the
log of the molecular weights of the subunits in kDa. A line of best fit was generated for the plotted data.
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𝑦 = −3.692𝑥 + 8.5281

4.45 = −3.6792𝑥 + 8.5281

−4.0781 = −3.6792𝑥

1.1084 = 𝑥

𝑥 = 𝐿𝑜𝑔 𝑀𝑊

1.1084 = 𝐿𝑜𝑔 𝑀𝑊

𝑀𝑊 = 12.84 𝑘𝐷𝑎

2C) Calculation of Band Size

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
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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

High Salt wash Lane (Figure 2A).

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

cause the molecular weight to be calculated as a lower weight.

Overall, the experiments demonstrated that Rubisco could be isolated using the proper

purification techniques such as ammonium sulfate precipitation and ion exchange

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
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because these proteins can now be studied, manipulated, and experimented with to gain a greater

understanding of their mechanisms.


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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,

L.M.C. (2014). Isolation and kinetic characterization of hydrophobically distinct populations of

form I Rubisco. Plant Methods, 10:17.

Salvucci, M.E., Werneke, J.M., Ogren, W.L., Portis, A.R. (1987). Purification and Species

Distribution of Rubisco Activase. Plant Physiology, 84: 930-936.

Spreitzer, R.J. & Salvucci, M.E. (2002). Rubisco: Structure, Regulatory Interactions, and

Possibilities for a Better Enzyme. Annual Reviews Plant Biology, 53:449-475.