Yana Kroytor

Lab Report 3

Lab 004

11/29/16

Cell Fractionation of Alfalfa Materials and Sucrose Gradient to isolate Chloroplasts and

Lysosomes

Introduction

Isolating the organelles requires the use of physical chemistry techniques, and those

techniques can range from the use of simple, gravity sedimentation, or ultracentrifugation of

fluorescent labeled organelles in computer generated density gradients. One method for

further purifying fractions is equilibrium density-gradient centrifugation, which separates

cellular components according to their density. This method also works well for resolving

lysosomes, mitochondria, and peroxisomes in the initial mixed fraction obtained by differential

centrifugation.1 It can further purify cell fractions obtained by differential centrifugation. Some

factors that affect particle movement are particle size, particle density, and solute

density/viscosity. Some research that was done in the 1920’s, Svedberg had done work with

colloids supported the theories of Brownian motion put forward by Albert Einstein and the

Polish geophysicist Marian Smoluchowski. During this work, he developed the technique of

analytical ultracentrifugation, and demonstrated its utility in distinguishing pure proteins one

from another.2

The energy of sunlight striking chlorophyll causes chloroplasts to synthesize sugars,

starches and free oxygen. The stroma of chloroplasts produces carbon fixation reactions known

as “dark reactions”. They will store energy in the form of adenosine triphosphate and

nicotinamide adenine dinucleotide phosphate molecules.3 The other organelle that will be

analyzed is the lysosome. Lysosomes are the main sites of digestion, that is the break-down of

structures, within cells. Recent research suggests that lysosomes are organelles that store

hydrolytic enzymes in an inactive state. The system is activated when a lysosome fuses with

another particular organelle to form a ‘hybrid structure’ where the digestive reactions occur

under acid (about pH 5.0) conditions. From this ‘hybrid structure’ a lysosome is reformed for re-

use.4 The experiment will analyze each of the fractions for optical density, protein content,

chloroplast content and acid phosphatase activity (lysosomes). Including the generalization of

spectrometry to analyze presence of green or yellow color.

Materials and Methods

Fractionation of Plant Materials

Approximately 15-20 g of alfalfa sprouts and 3 mL of grinding media were added (0.4 M

sucrose, 0.165 M Tris-HCl buffer at pH 7.5, 0.01 M KCl, 0.01 M MgCl2, 0.01 M EDTA adjusted to

pH 7.5, 0.01 M dithiothreitol) to become a fine paste in the mortar. During the grinding process,

20 mL of grinding media was added gradually which would then turn the sprouts into brei. Two

layers of cheesecloth was placed over a 150 mL beaker. The brei was filtered through the

cheesecloth into the beaker, which was then transferred into a polypropylene centrifuge tube,

and the tube will be in the centrifuge for 10 minutes at 270 xg (1,500 RPM in the Beckman JA-

17 Rotor) to remove unbroken cells and large debris.

When the 10 minutes were done, the supernatant was removed into a clean, cold

centrifuge tube and ran it again through the centrifuge for another 30 minutes at 10,800 xg

(10,300 RPM for JA-17 Rotor). After the 30 minutes have concluded, the supernatant was

carefully removed from the formed pellet. The pellet was then resuspended in 2 mL of grinding

medium and was held in ice for further analysis. The supernatant was then discarded. Using a

Pasteur pipette, the 2 mL layer was carefully layered on top of the sucrose linear gradient.

Fractionation of Sucrose Gradient and Gradient Detachment

A linear 33-60% sucrose gradient in a polyallomer ultracentrifuge tube was prepared. To

the bottom of the centrifuge tube, 5 mL of the 60% sucrose was added. The controlling of the

connection between the two chambers was controlled by a thumbscrew. In the right chamber,

15 mL of the 60% sucrose was added and in the left, 15 mL of the 33% sucrose was added. The

entire gradient was placed on a magnetic stirrer and a small magnet was inserted in the right

side. The stirrer was turn to a medium level stirring. After the sucrose gradient was formed, the

2 mL pellet were layered on top of the sucrose.

Each tube that was given, were weighed in accordance to them being equal. The two

tubes were equal in weight and then added to a SW32 rotor that was then centrifuged at 4°C

for four hours at 25,000 RPM. The tubes were divided into 20 fractions, those fractions

contained 1.5 mL of the samples by using a micropipette to transfer. A 2.0 mL syringe was

attached was inserted with an 18-gauge needle and fractions of 1.5 were carefully removed

until all of the supernatant was removed.

Analysis of Optical Density and Absorbance

The optical densities of the fractions were analyzed at 540 nm and 673 nm (in order to

analyze chloroplasts) by using Vernier Logger Pro software and a SpectroVis

spectrophotometer. Each fraction had samples of 1.0 mL using a blank consisting of 33%

sucrose.

Bradford Protein Assay

In 20 individual Spec20 tubes, fraction samples of 100 μL were added. Final

concentrations of 1500, 750, 375, and 187.5 μg BSA/mL were prepared using a bovine serum

diluted with 0.15 M NaCl to produce 200 μL of standards. In each of the individual Spec20

tubes, 100 μL of each concentration of the protein standards were added, including an extra

tube that is a blank using 100 μL of 0.15 M NaCl. After all of the Spec20 tubes were prepared,

an additional 3.0 mL of Bradford Reagent were added in the fraction samples, blank, and the

protein standards. The tubes were covered in parafilm which was left to sit for 5 minutes. Using

the Bradfard Reagent as the blank, the tubes were then analyzed for their absorbance at 595

nm.

Utilizing the molar extinction coefficient (C=A/ε) was calculated from the known

standard concentrations and their absorbance’s. The protein content was plotted in a fraction

number after deciding the amount of protein in each sample utilizing that exact molar

extinction coefficient.

Analysis of Acid Phosphatase Activity

Using a micropipette, a 100 μL of each fraction was transferred into a Spec20 tube. In

each of the individual tubes, 0.5% sodium cholate (200 μL) was added to lyse the membrane,

including a mix of 0.5 M acetate buffer (5.0 pH) of 200 μL. Before the tubes were added in the

water bath that stays at a constant temperature of 37 °C for 10 minutes, another mixture was

added to the tubes of 10 Mm p-nitrophenyl phosphate (500 μL). In order to stop the reaction o

keep proceeding, in each tube, 3.0 mL of 0.5 M NaOH was added and then inverted. To analyze

the absorbance of each tube, it was measured at 405 nm using regular water as a blank. The

products formed were determined by utilizing the Beer-Lambert law and extinction coefficient

of 18.2 mM-1.

Results

The results for this experiment, the different assays and activities executed will identify

the presence of lysosomes by the activity of acid phosphatase in each fraction.

The first assay was the optical density assay which observes each fraction to see which

one has the most particles. The particles stopped the light from transmitting and gives the

absorbance levels. The particles

were assumed to be pieces of

organelles and pieces of the cell

based on the data. The larger

particles were filtered out by

earlier sections of the experiment.

In Figure 1. there were clear peaks

at fraction numbers 2, 3, 8, and

small peak at 17 because their peak

levels were higher relative to the

ones around it. These peaks

correspond to density of sucrose which means that the absorbance can be analyzed with the

density of sucrose to fully understand the density of the particles. The fact that the absorbance

Figure 1. The comparison of absorbance at 540.2 nm and sucrose density to fraction number in optical density. The density of the sample will be referenced to

the absorbance data.

gives the knowledge of density, then in the other graphs, the densities can be analyzed to

determine specific organelles. Also, in the graph there was a general increase after fraction 10

for absorbance. The sucrose gradient was linear and would decrease because the gradient had

more of a concentration in the bottom and less concentrated sucrose gradient as it went up the

tube. The absorbance at 540.2 nm shows the relative amount of particles that refracts light.

More analysis of the optical density shows a more exact relevance of the fraction

number and the absorbance at 673.4 nm. This graph specifically will show the presence of

chloroplasts. There are two

sections to this first assay and

the first section was to

determine the density relative to

the absorbance whereas the

second section shows the

relative correspondence of

absorbance and the fraction

number. Figure 2 shows a large

peak at fraction 2, medium

fraction peaks at 8, fraction 11,

fraction 17, and a smaller peak

at fraction 21. This shows that

those fractions had high concentrations of chloroplasts. The other areas have chloroplasts but

Figure 2 The comparison between the absorbance of each fraction number based on the optical density activity that will look at the presence of chloroplasts. The absorbance that was measured was 673.4 nm and there were strong distinct peaks at fraction 2, fraction 8, fraction 11, and small fractions 17 and 21. In Figure 1, it showed the high concentrations of chloroplasts in each fractions which then is relevant to the sucrose densities at 1.28 g/mL, 1.17 g/mL, and 1.15 g/mL. The distinct peaks show high concentrations of pieces of the cell that absorbed at the wavelength of 673.4 nm.

not as concentrated as the other clear, large peaks. It can also be pieces of chloroplasts instead

of a small amount of concentrations.

The second assay was the Bradford Protein assay and that shows the protein

concentration relative to the fraction number. It shows which fraction has the most amount of

proteins. The figure is shown as a Beer-Lambert plot that gave an extinction coefficient. The

relative fractions that show the most proteins are fractions that are gradually increasing. The

differential peaks and divots in the graph are responsible for the different areas in the sucrose

gradient. The sucrose

gradient decreases in

concentration as it goes

up the tube, though

throughout the tube,

there may be areas that

have no protein

(possibly gaps in

between each protein)

that will not show up

on the Beer-Lambert

plot. In accordance to

the fact of the gaps in the sucrose gradient, that gives the graph those decrease divots. In

Figure 3, it shows the gradual increase by each fraction and the clear peaks at fraction 2,

fraction 8, and then it increases with each peak with little decreases. The proteins are the

Figure 3 The comparison of protein concentrations of each fraction number based on the Bradford Assay activity. The Beer-Lambert plot shows the presence of lysosomes in each fraction.

enzymes which are acid phosphatase, which is shown by the concentration of the protein

concentration. The earlier peaks in the plot can possibly be a small amount of chloroplasts, or

possible pieces of chloroplasts.

The last assay was the acid phosphatase activity which shows the relevance of pNP and

each fraction number. Acid phosphatase is an enzyme that shows lysosomes on the graph. The

substrate is the pNP and it is combined with the acid phosphatase which will then measure the

activity which is then

shown on the graph. In

figure 4, there is a clear

increase of acid

phosphatase activity and

each fraction goes up. The

decrease divot around

fraction 21 is not a clear

indication on why that

happened in the first

place. That fraction did

not have acid phosphatase activity but the next fraction allowed the acid phosphatase to keep

that activity at the same pNP concentration. This graph has relevance to Figure 2, because they

both show the fractions that either have chloroplast (Figure 2) or lysosomes (Figure 4). Both of

the graphs almost look like an inverse of each other.

Works Cited

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Fraction Number

[pNP

] (m

M)

Figure 4 The comparison of pNP concentrations of each fraction number based on the Acid Phosphatase activity. The reaction was recorded at an absorbance of 405 nm. The specific activity of the acid phosphatase was 8.968 x 10-5 units/mg. The substrate in this assay was pNP which were in each fraction. The specific peaks that accounted for the pNP concentrations are fractions 8, fraction 11, and fraction 16. There was a distinct downward peak around fraction 21.

1 Lodish, Harvey. "Purification of Cells and Their Parts - Molecular Cell Biology - NCBI Bookshelf."

National Center for Biotechnology Information. U.S. National Library of Medicine, 1970.

Web. 26 Nov. 2016.2 "DNA-RNA-Protein." DNA-RNA-Protein. N.p., n.d. Web. 26 Nov. 2016.3 "Chloroplast." Chloroplast | Structure, Chloroplast Function | Biology@TutorVista.com. N.p.,

n.d. Web. 27 Nov. 2016.4 "Lysosome." British Society for Cell Biology. N.p., n.d. Web. 27 Nov. 2016.