Josephine Moore

BCOR Lab Report

Introduction

Proteins play many important roles in the human body. Proteins are organic compounds

that have one or more long chains of amino acids which are essential to all living organisms.

Every cell in the body has proteins vital for maintaining proper structure, function, and

regulation of our tissues and organs. Enzymes, a subset of proteins, are biological molecules that

play a crucial role in catalyzing and regulating various biochemical reactions within living

organisms. Lysozyme is an enzyme found in various biological systems, including tears, saliva,

mucus, and egg whites. This enzyme is an important component of the body’s immune system as

it results in the destruction of bacterial cells and is the first line of defense against bacterial

infections in the eyes, mouth, and respiratory and gastrointestinal tracts. Thereby useful in

preventing infections and maintaining overall health.

In this experiment, chicken egg white lysozyme activity will be tested at various salt

concentrations, 100mM NaCl and 750mM NaCl, at different pH’s and temperatures. Through the

experiment, the expectation is to measure lysozyme activity through absorbance of wavelength

in order to find the optimum temperatures and pH for lysozyme activity. Then, using the results

of the experiment, it will be determined in which applications they can be used and how. Overall,

the experiment to be conducted will answer these hypotheses; when the highest concentration of

salt (750mM) is added to lysozyme activity, pH and temperature increases. When the

temperature is lower, then activity of the egg white lysozyme decreases.

Materials and Methods

Step 1: Purify the Lysozyme

Josephine Moore
BCOR Lab Report

The first step was to purify the lysozyme. To do this, 10ml of a filtered egg white was

mixed with 60ml of 25mM glycine buffer to dilute in a beaker. Then, 2ml of the filtered egg

white was poured into the column that contained 2ml of 25mM glycine buffer while saving

effluent. Column was then flushed using 2ml of 25mM glycine and saved to be eluted using 2ml

of 100mM NaCl, 25mM glycine which was also saved. Then, the column of 2ml of 100mM

NaCl, 25mM glycine buffer was flushed and saved to be eluted with 2ml of 750mM NaCl,

25mM glycine buffer, also saved. Column was rechanged through a wash with 2ml of 100mM

NaCl, 25mM glycine, then also 2ml of 25mM glycine. The amino acid sequence of three main

proteins found in chicken egg whites: ovalbumin, ovotransferrin, and lysozyme were used in

determination of which of the collected fractions these proteins were present.

Step 2: Identify Protein Fraction

Next, the protein fraction with enzyme activity was identified. The wavelength was set to

450nm on the spectrophotometer and zeroed using phosphate buffer. 5 large test tubes were

labeled; flow through fraction, flush 1, 100mM NaCl fraction, flush 2, 750mM NaCl fraction

(respectively). 2ml cell wall suspension of dried bacterium Micrococcus lysodeikiticus as a

control was added to each tube and the initial absorbance was recorded. 100ul of sample fraction

was then quickly added to each tube while reaction timing began until 3 minutes for the final

absorbance recording. Then, the baseline activity rate was determined by first zeroing the

spectrophotometer and adding the control to a test tube for its initial absorbance reading. Again,

100ul of active sample fraction was added to tube and timing began; absorbance was recorded

every 30 seconds for 3 minutes. Following this, the effects of temperature and pH on enzyme

activity testing began. Initial absorbance was recorded and pH was tested with the

spectrophotometer using same procedure as for baseline activity rate now with different pH’s (5,
Josephine Moore
BCOR Lab Report

6, 7 and 8) as well as temperatures (30°C, 40°C, 50°C and 60°C). The only difference for pH was

to use specific pH in place of cell wall suspension control. The only difference with temperature

is that the cell wall suspension was incubated to specific temperature prior to adding the active

fraction and after each reading the tube was placed in the heat block (except for the active

fraction stock solution).

Step 3: Determining Protein Concentration

Now, protein concentration was determined using Bradford Assay. 3mL Bradford reagent

was added to each, along with 100uL, 90uL, 75uL, 50uL, and 0uL buffers, then with 0uL, 10uL,

25uL, 50uL, and 100uL BSA solution was all added to 5 test tubes respectively and labeled. The

amount of protein (ug) as well as absorbance at 595nm was calculated and recorded. Then, the

unknown samples were prepared for analysis by adding 3mL Bradford reagent, 0uL buffer, and

100uL protein fraction to the flow through, 100mm, and 750mm fraction. Here, absorbance at

595nm was recorded. A blank fraction with 3mL Bradford reagent, 100uL buffer, and 0uL

protein fraction was recorded as a control. The protein concentration (ug/uL) and volume needed

for 6ug was then calculated.

Step 4: SDS-PAGE Gel Electrophoresis

Finally, SDS-PAGE gel electrophoresis technique was used to separate molecules based

on their molecular weight and/or charge when placed in an electrical field. First, the gel was

prepared using a pre-cast with any kD polyacrylamide gel. Once the gel was prepared, 1X

Running Buffer (TRIS/SDS Buffer) was poured into the center chamber and 1X Running Buffer

was poured into the exterior chamber to cover the gels up indicating 4 gels on the gel running

apparatus. Now, the samples were prepared and loaded using ~6ug of each sample including
Josephine Moore
BCOR Lab Report

flow through, 100mM NaCl and 750mM NaCl. A Loading Sample Buffer (3X LSB) and water

was used to bring the volume up to ~20uL and samples were set up in Eppendorf tubes. The

tubes were micro-centrifuge to ensure all liquid went to the bottom of the tube. All samples were

then boiled for 3 minutes in the heat block and after were micro-centrifuge once more. The

sample was then placed in the gel and ran at 400V for 14 minutes. Then, the gel was rinsed with

distilled water for 3 minutes. The water was discarded and 20mL of Coomassie blue stain was

added. After 45 minutes the results were checked and left overnight if needed.

Results

Absorbance of Cell Wall Suspension at

450nm
0 minutes 3 minutes
1
0.8
Absorbance

0.6
0.4
0.2
0
Flowthrough Flush 1 100 mM NaCl Flush 2 750 mM NaCl
Fraction

Figure 1 Absorbance, ranging from 0 to 1, of cell wall suspension at 450nm for 5 different

fractions, flowthrough, flush 1, 100mM NaCl, flush 2, and 750mM NaCl, measured at 0 and 3

minutes
Josephine Moore
BCOR Lab Report

Lysozyme Activity at Different Temperatures

2
1.8
1.6
1.4
1.2
Absorbance

1
0.8
0.6
0.4
0.2
0
0 20 40 60 80 100 120 140 160 180 200
Time (sec)

Room Temp. 30°C 40°C 50°C 60°C

Figure 2 Lysozyme activity measured in absorbance (wavelength) at different temperatures

(30C, 40C, 50C, 60C, and room temp) over time (seconds)

Slopes for Temp (0-60sec)

30sec y=-0.0081x+1.8608
40sec y=-0.0117x+1.7428
50sec y=-0.0087x+1.4558
60sec y=-0.0098x+1.2917

Table 1 Slopes for temperature (30C, 40C, 50C, 60C, and room temp) measured from 0 to 60

seconds as represented by Figure 2 graph

Josephine Moore
BCOR Lab Report

Lysozyme Acti vity at Diff erent pHs

pH 5 pH 6 pH 7 pH 8 Baseline
2
1.8
1.6
1.4
1.2
Absorbance

1
0.8
0.6
0.4
0.2
0
0 20 40 60 80 100 120 140 160 180 200
Time (sec)

Figure 3 Lysozyme activity measured in absorbance of wavelengths in nm tested at different

pH’s ranging from 5-8

Slopes for pH (0-60sec)

pH 5 y=0.0005x+0.8817

pH 6 y=0.0015x+0.1483

pH 7 y=-0.0082x+1.715

pH 8 y=-0.001x+1.1633

baseline y=0.0113x+0.9533

Table 2 Slopes represented from graph of Figure 3 for the different pH’s (5, 6, 7, 8, and

baseline)
Josephine Moore
BCOR Lab Report

Protein Amount (ug) and Absorbance (595nm)

1

0.9 f(x) = 0.00922117202268431 x

R² = 0.99753564105948
0.8
Absorbance (595nm)

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0
0 20 40 60 80 100 120
Protein Amount (ug)

Figure 4 Protein amount measured in ug was tested with absorbance at 595nm ranging from 0

to 1 absorbance
Josephine Moore
BCOR Lab Report

Protein Concentration (ug/uL) for Unknown Protein

Samples
1.2

1
Protein Concentration (ug/uL)

0.8

0.6

0.4

0.2

0
Blank Flow Through 100mM NaCl 750mM NaCl
Sample

Figure 5 Protein concentration measured in ug/uL for protein samples flow through, 100mM

NaCl, 750mM NaCl, and a blank as control

Josephine Moore
BCOR Lab Report

Figure 6 A gel electrophoresis photograph represents ovotransferrin and ovalbumin in wells 1-

7, a control in well 9, and a ladder for comparison in well 10

Josephine Moore
BCOR Lab Report

Figure 7 A second gel electrophoresis photograph represents lysozyme activity from the

750mM fractions in wells 1, 2, 5, and 7, with a lysozyme control in well 9 and a ladder for

comparison in well 10
Josephine Moore
BCOR Lab Report

Absorbance, ranging from 0 to 1, of cell wall suspension measured at 450nm wavelength

was experimentally found for 5 different sample fractions at 0 minutes and 3 minutes.

Absorbance at 0 and 3 minutes was typically close to equal with 3 minutes slightly lower, except

for the 750mM NaCl fraction. Flowthrough sample was found to be at the highest absorbance for

both 0 minutes and 3 minutes at slightly above 0.8. Flush 2 was found at the second highest at

slightly below 0.8 absorbance. Then, 750mM NaCl at 0 minutes was found at 0.6 absorbance,

but dropped to 0 at 3 minutes. Following was 100mM NaCl in between 0.4 and 0.6 absorbance

for both 0 and 3 minutes. Finally, flush 1 was found between 0.2 and 0.4 absorbance. (Figure 1)

Enzyme activity of the lysozyme, measured in absorbance of wavelengths, decreased as

time increased for all temperatures tested; up to 200 seconds was observed. The lower

temperatures, 30ºC being the lowest, had the highest absorbance throughout the entire timed

experiment. Higher temperatures, including 60ºC as the highest, was at the lowest absorbance for

the entire timed experiment. (Figure 2)

Enzyme activity of lysozymes at different pH’s, measured in absorbance over 200

seconds, remained relatively constant with minimal fluctuation. Starting at a pH of 5, lysozyme

activity decreased slightly from start, 0 seconds about 1nm absorbance to 0.5nm absorbance at

around 175 seconds. For a higher pH, as tested at 8pH, lysozyme activity remained relatively

constant starting at around 1.2nm at 0 seconds to about 1.2nm at 175nm. However, pH of 6

measured in at around 0.1nm absorbance at 0 seconds and ended at around 0.25nm absorbance at

around 175 seconds. A pH of 7 began at 1.75nm absorbance at 0 seconds, to 0.9nm absorbance at

175 seconds. The baseline pH remained highest of all pH after about 35 seconds to 175 seconds.

(Figure 3)
Josephine Moore
BCOR Lab Report

The amount of protein measured in ug has a positive correlation to absorbance measured

in wavelength at 595nm. As protein amount increases, absorbance in wavelength also increases.

Protein amount ranged from 0ug to 100ug and absorbance ranged from 0 to 1 at 595nm. (Figure

4)

Protein concentration measured in ug/uL ranging from 0 to 1.2 ug/uL was tested for each

sample, including flow through, 100mM NaCl, and 750mM NaCl. Flow through sample

represented the highest protein concentration at around 1.15ug/uL, next 100mM NaCl at around

0.15ug/uL, and finally 750mM NaCl at around 0.125ug/uL. Blank was included as a control.

(Figure 5)

Lysozyme marker did not show up in first gel, but ovotransferrin and ovalbumin showed

up in fractions 100mM and flowthrough in wells 1-7. A lysozyme control is represented in well 9

and a marker of all potential protein sizes as a ladder in well 10. (Figure 6)

The lysozyme at the bottom of the block is visible from the 750mM fractions added to 1,

2, 5, and 7 with a lysozyme control sample in well 9 and a marker of all potential protein sizes as

a ladder in well 10. (Figure 7)

Discussion

To discuss the results, the major factors include the trials regarding pH and temperature.

Lysozyme activity increased with increasing pH as shown in figure 3 and decreased as

temperature increased as shown in figure 2. Also, the higher the salt concentration brought a

higher absorbance, though the flowthrough sample was the highest and as protein concentration

increased, as did absorbance. Through these results, the hypothesis can be confirmed. The

hypothesis states if the highest concentration of salt (750mM) is added to lysozyme activity, then
Josephine Moore
BCOR Lab Report

the pH and temperature will increase. The data mostly supports this hypothesis as pH and

temperature were tested with this salt concentration (750mM) and pH was shown to increase in

figure 3, however, as higher temperatures were tested, lysozyme activity decreased over time

shown in figure 2. Because salt concentrations increase the number of ions in the solution and

alters the shape of the enzyme, the lysozyme activity for a higher pH should have increased as it

did. Also, because enzymes are proteins, the increase of ions decreases the freezing point in

water and therefore increase enzyme activity. With a lower concentration of salt, this ability

would decrease, however too high and an enzyme will denature, making the enzyme unstable.

So, it is possible that using 750mM salt concentration was too high, creating a decreased

enzymatic activity when testing temperature. Though, this is unlikely as there was still lysozyme

activity at this salt concentration and due to the positive correlation between a higher salt

concentration and pH. The hypothesis of if the temperature is lower, then activity of the egg

white lysozyme will decrease, is not supported by the data. While higher temperatures did not

increase lysozyme activity, all temperatures decreased together, however the data does not

support the hypothesis because the lower temperatures remained at a higher activity than the

higher temperatures. This could be due to lysozymes having an optimal temperature with

activity. Meaning, it may be true that with this salt concentration, the lowest temperature was

actually the optimal temperature and moving to higher temperatures began to denature the

enzyme, slowing activity. To test this further, more trials with higher and lower temperatures at

all the salt concentrations would be helpful in further determination of activity. Also, failure to

use equipment properly, mathematical errors, etc.. could have come into play in hindering

results. As for the gel electrophoresis machine specifically, the results came out foggy and

unclear due to an electrical error with the machine itself, which caused the hinderance in results.
Josephine Moore
BCOR Lab Report

Based on the experiment with Miyazaki, the optimal temperatures for the fish lysozymes were

increased during warmer months and more effective and their activity decreased when

temperature decreased. Similarly, the temperature for the chicken egg white lysozyme did

decrease with temperature, but never reached an optimal temperature. Furthermore, these results

have implications beyond the scope of the experiment in the medical field. Lysozyme tests

measure the levels of an enzyme that supports the body’s immune system. So, in testing the

optimal lysozyme activity at different temperatures and pH’s can aid in diagnosis of certain

immune disorders, such as leukemia, sarcoidosis, and infections like tuberculosis. Elevated

levels of lysozyme activity can be treated with temperature or pH additives that can lower

lysozyme activity to a normal rate to ensure disorders do not increase to kidney damage and

more life-threatening chronic diseases. Conversely, lower lysozyme activity levels can be treated

with proper temperature or pH in order to increase activity to an optimal level. Also, by testing

lysozyme activity of certain food products, it may be possible to use create optimum lysozyme

activity as treatment for these diseases through food or pharmaceuticals. Overall, while the

results did not yield perfect results, they created a basis for more research and trials that could

have major implications in the medical, pharmaceutical, and food industries.

Josephine Moore
BCOR Lab Report