Chemistry Research

ABSTRACT

Lysozyme, discovered in 1922 by accident by Alexander Flemming, is a globular protein containing 129 amino acids. This enzyme was later found in bodily secretions, plants, and most abundantly in egg whites. As part of this project, a new method of extracting lysozyme from egg whites was developed. The activity and therefore the amount of lysozyme extracted from both white and brown eggs was measured through absorbance assays using Micrococcus lysodeikticus cell walls and Michaelis-Menton enzyme kinetics. It appears that more lysozyme is found in brown eggs compared to white eggs.

INTRODUCTION

Lysozyme is a globular protein containing 129 amino acids that was discovered in 1922 by Alexander Flemming by accident (5). He placed some of his nasal drippings onto a petre dish containing bacteria and noticed that the cells were lysed. This enzyme was later found in other bodily secretions, plants, and most abundantly in egg whites. Unfortunately, Flemming also discovered that lysozyme was not the wonder antibiotic that he was searching for.

Lysozyme's active site cleaves the bond between the N-acetylmuramic acid (NAG) and N-acetylglucosamine (NAM) monomer units of the peptidoglycan found in the cell walls of some bacteria. The relative hydrolytic rate of lysozyme is comparably slow, but because of the high osmotic pressure within bacteria cells, only a few "nicks" in the cell wall cause the bacteria cell to rupture (3).

Since lysozyme's discovery, it has been important to the scientific community. It was the first enzyme to have its entire three-dimensional structure determined with the aid of X-ray crystallography in the 1950's (5). Lysozyme's mechanism of lysing bacteria cell walls helped scientist understand how enzymes and their substrate fit together and release products. Shugar developed an assay to measure the activity of lysozyme through absorbance measurements (6). The substrate in these assays is reconstituted freeze-dried Micrococcus lysodeikticus bacteria cell walls. A solution containing the cell walls absorb at 450 nm and as lysozyme works on breaking the M. lysodeikticus cell walls into smaller and smaller pieces, the absorbance decreases. Using the Shugar methods and Michaelis-Menton enzyme kinetics, lysozyme activity can be quantified in different situations. The reconstituted freeze-dried lysozyme used in Shugar assays has been commercially purified from egg whites because this is a very accessible and very abundant source.

One of the goals of this experiment was to develop an easier method of lysozyme isolation that could be completed in college laboratories. Another goal was to determine if caged-commercial chicken's eggs lysozyme levels differ from free-range farm egg lysozyme levels. The commercial eggs used were white and the farm eggs used were brown. According to the Egg Nutrition Center, there are no taste or nutritional difference between brown and white eggs (2). But perhaps the living conditions and stress levels of the chickens have some effect on the amount of lysozyme placed in the eggs.

MATERIALS

Instruments:

pH meter
Varian DMS 100S UV/Visible Spectrophotometer, test tubes
Sonicator
Stirring plates
Vacuum filter assembly
Micropipette

White eggs, from local grocery store
Brown eggs, from Janet Rasmusson's flock
Freeze-dried lysozyme, from Sigma Chemical Co.
Freeze-dried Micrococcus lysodeikticus cell walls, from Sigma Chemical Co.
Amberlite IRC-50 resin beads, from Sigma Chemical Co.
KH₂PO₄ and K₂HPO₄, from Fisher Scientific

METHODS

Preparation of Reconstituted Lysozyme Stock Solution:

Placed 4 mg Sigma lysozyme in test tube and added 4 mL of distilled water. After stirring, 400 uL aliquots were put in smaller test tubes, corked and placed in freezer until needed.

0.1 M Phosphate Buffer Solution ( pH 7.0):

13.618 g of KH₂PO₄ placed into 1 L volumetric flask, distilled water added up to the mark. In another 1 L volumetric flask, 17.518 g of K₂HPO₄ is added then diluted up to the mark with distilled water. With the aid of a pH meter, approximately equal parts of the two solutions are added to make a buffer solution of pH 7.0.

0.5 M Phosphate Buffer Solutions (pH 6.5 & 8.0):

Into a 1 L volumetric flask is added 68.09g of KH₂PO₄ and enough distilled water to reach the mark. In a separate 1 L volumetric flask, 87.59 g of K₂HPO₄ is added then diluted up to the mark with distilled water. For the buffer pH 6.5, approximately 100 mL of KH₂PO₄ is mixed with 650 mL of K₂HPO₄ as the pH is monitored with a pH meter. The 8.0 pH buffer is an approximate mixture of 240 mL of KH₂PO₄ and 650 mL of K₂HPO₄ also monitored by a pH meter.

Protocol:

Isolation of Egg White

1. Recorded mass of whole egg
2. Separated egg white and placed in 250 mL beaker
3. Sonicated egg whites in beakers for 1/2 hour
4. Added 7.5 mg Amberlite resin beads to beaker after removal of egg white from sonicator
5. Stirred egg white/resin beads at slow speed for 1 hour on magnetic stirring plate
6. Washed egg white from resin beads with three 50 mL portions of Pi(K +) 6.5 pH buffer solution
7. Swirled, let settle for approximately 30 seconds, decant off excess with minimal loss of resin beads
8. Use disposable 1 mL pipette to suck off excess buffer/egg white
9. Add 10 mL of phosphate pH 8.0 buffer solution
10. Let resin/pH 8.0 buffer stand at room temperature uncovered for needed time (5, 15, 30, 45 min etc)
11. Vacuum filter buffer/lysozyme solution from resin beads
12. Cover and refrigerate lysozyme solution for later analysis

Standard Curve Assay for Sigma Lysozyme

1. Prepare M.lysodeikticus (Ml) solution: Combine 9 mg Ml cells with 30 mL water, cover and stir
2. Prepare diluted Sigma Lysozyme soluiton: Combine 10 uL of stock lysozyme solutions (1mg/1mL) and 990 uL distilled water
3. Spectrometric tubes (path length 1 cm) are labeled 1-5
4. 2.9 mL of Ml solution is added to each tube
5. 0, 100, 150, 175, or 195 uL of phosphate pH 7.0 buffer is added to tubes 1-5, respectively
6. Tube 1 absorbance at 450 nm is measured, then 200 uL of diluted lysozyme solution is added, absorbance measured every 15 seconds for 2 minutes
7. Tube 2, ditto except 100 uL of diluted lysozyme added
8. Tube 3-5, ditto, except 50, 25, and 5 uL of diluted lysozyme added
9. Absorbancies recorded and plotted verses time for each concentration of lysozyme

Activity Assays for Isolated Egg White Lysozyme

1. Prepare M. lysodeikticus (Ml) solution: Combine 9 mg Ml cells with 30 mL water, cover and stir
2. Spectrometric tubes (path length 1 cm) are labeled
3. 2.9 mL of Ml solution is added to each tube
4. Tube 1 absorbance at 450 nm is measured, then 200 uL of isolated lysozyme solution is added, absorbance measured every 15 seconds for 2 minutes
5. Repeated as needed for different isolations

RESULTS

From the graph of absorbance verses time of lysozyme lysing M. lysodeikticus cell walls, the initial velocity can be calculated from the slope. Then, these initial velocities of each lysozyme concentration were plotted verses lysozyme amount (in ug). According to Michaelis-Menton enzyme kinetics theory, a double-reciprocal plot of 1/initial velocities and 1/enzyme concentration will give a linear relationship (see Graph 1). The resulting straight-line equation (Equation 1) was used to determine the amount of lysozyme extracted from various egg whites from their initial velocities.

Equation 1: Straight-line Equation for ug lysozyme

Y = 11.880 X + 0.750, Y = 1/ Vo
X = 1/ug lysozyme
Rearranged:
11.880/((1/Vo)-0.750) = ug lysozyme

Table 1 summarizes the data for Graphs 2 and 2a. These Graphs look at the effect of extraction time of the resin beads with attached lysozyme and the pH 8.0 phosphate buffer. Graph 2 looks at the activity of lysozyme over time while Graph 2a shows just the initial velocities.

Table 1: Brown vs. White eggs with 30 and 15 min Extraction with pH 8.0 buffer

Time (min)	Brown, 30 min	Brown, 15 min
0	0	0
0.1667	0.07	0.053
0.333	0.12	0.0655
0.5	0.18	0.0875
0.667	0.23	0.106
0.833	0.28	0.123
1	0.322	0.14
Vo (slope from Graph 2a)	0.42	0.318
Lysozyme Extracted, ug	7.2841	4.9610
Time (min)	White, 30 min	White, 15 min
0	0	0
0.25	0.036	0.0355
0.5	0.046	0.0535
0.75	0.0565	0.0705
1	0.0665	0.088
Vo (slope from Graph 2a)	0.144	0.142
Lysozyme Extracted, ug	1.9178	1.8880

Graphs 3 and 3a compare the effect of extraction time on the amount of lysozyme extracted from white eggs. Table 2 summarizes this data. Graph 3 shows the lysozyme activity over time, while Graph 3a shows the initial velocities from which the amount of lysozyme can be calculated using Equation 1.

Table 2: Comparison of Extraction Time with pH 8.0 buffer for White eggs, 4 days at 11.5 ^oC

Time (min)	White, 5 min	White, 15 min	White, 30 min	White, 45 min
0	0	0	0	0
0.25	0.0525	0.0355	0.036	0.038
0.5	0.074	0.0535	0.046	0.046
0.75	0.097	0.0705	0.0565	0.3715
1	0.117	0.088	0.0665	0.061
Vo (slope from Graph 3a)	0.21	0.142	0.144	0.152
Lysozyme Extracted, ug	2.9612	1.8880	1.9178	2.0381

Table 3 contains the data for Graphs 4 and 4a. Graph 4 compares the effect of pH 8.0 buffer incubation time for Brown eggs over time. The initial velocity is seen in Graph 4a.

Table 3: Comparison of Extraction Time with pH 8.0 buffer for Brown eggs, 1 days at 11.5 ^oC

Time (min)	Brown, 15 min	Brown, 30min	Brown, 45 min
0	0	0	0
0.1667		0.06	0.052
0.25	0.057
0.333		0.114	0.077
0.5	0.0805	0.168	0.104
0.667		0.219	0.1285
0.75	0.104
0.833		0.266	0.1525
1	0.1265	0.308	0.177
Vo (slope from Graph 4a)	0.228	0.360	0.312
Lysozyme Extracted, ug	3.2674	5.8586	4.8389

Finally, Graphs 5 and 5a demonstrate the effect of time on lysozyme activity when kept in the refrigerator for one month. Graph 5a looks specifically at the initial velocity. All the data for these graphs are found in Table 4.

Table 4: Deactivation of Lysozyme over time at 11.5 ^oC

Time (min)	8 days old	30 days old
0	0	0
0.25	0.0355	0.015
0.5	0.0535	0.023
0.75	0.0705	0.0305
1	0.088	0.019
Vo (slope from Graph 5a)	0.142	0.060
Lysozyme Extracted (in ug)	1.8880	0.7464

DISCUSSION

Lysozyme was first introduced to the author the summer of 1998 in a biochemical laboratory at Bowling Green State University (BGSU). Under the direction of Dr. Brecher, an enzyme kinetics study was done on the effect of chlorambucil pre-incubation with lysozyme. At Heidelberg College in the fall semester of 1998, the author's microbiology research project (under the direction of Dr. Susan Carty) also involved lysozyme. She placed egg white and saliva on bacteria cultures to observe the effects of the lysozyme within these substances on bacteria growth. Finally as her senior honors project in the spring semester of 1999, the author and her mentor Dr. Dan Esterline developed a procedure to extract lysozyme from egg whites.

A literary search produced an article by Li-Chan et al who developed an extraction method using cation exchange column chromatography (4). The initial plan was to duplicate the Li-Chan procedure and then test the activity of the extracted lysozyme with the skills learned at BGSU. The type of resin beads that Li-Chan used was no longer available for purchase; therefore a substitution was made. The unavailable Dulite C-464 resin was very similar to Amberlite IRC-50 resin that Heidelberg College Chemistry Department already had in stock. These resin beads were composed of many monomer units linked together, the only difference being a hydrogen vs. methyl substituent (see Figure 1). Besides changing the resin, the very viscous egg whites did not flow by gravity or even under balloon pressure through the glass columns that were available, so an open-batch method was employed. The time of sonicating egg whites (a procedure employed to decrease viscosity), the amount of resin beads added, and the extraction time period with the phosphate buffer were all optimized resulting in the method described earlier (see page 4).

Figure 1: Difference between Dulite and Amberlite resins

Duolite C-464 Monomer: Acrylic Acid
Amberlite IRC-50 Monomer: Methacrylic Acid

In order to determine whether the described method was successful in extracting active lysozyme, initial tests were run. Initial absorbance tests at 280 nm showed that lysozyme was indeed present in the pH 8.0 buffer (4). Other absorbance tests at 450 nm demonstrated lysozyme activity because lysozyme was lysing the cell walls of Micrococcus lysodeikticus (6). After the absorbance test showed that the method was indeed extracting lysozyme from egg whites, the amount of lysozyme in each of the extractions was determined. A standard curve of purchased lysozyme activity on Micrococcus lysodeikticus (Ml) cell walls was obtained using the method described above. Employing Michaelis-Menton enzyme kinetics, a plot of the inverse initial velocity verse the inverse of lysozyme amount produced a linear relationship and a usable equation for determining the amount of lysozyme extracted from each egg white (see Graph 1 and Equation 1).

Brown and white eggs were used to observe any difference in the amount of lysozyme because of eggshell color. According to the Egg Nutrition Center, no nutritional differences are seen between brown and white eggs (2). The extraction time with the pH 8.0 buffer, the process used to remove lysozyme from the resin, was a variable studied. The effect of varying extraction times at room temperature was studied to determine the optimal extraction time.

Graphs 2, 3, 4, and 5 all show how the extracted lysozyme initially has more activity against the Ml cell walls and then the lysing ability levels off over time. Such enzyme kinetics results are typical. At first all lysozyme molecules are available to bind to substrate; but as the enzyme (lysozyme) activities begin to "fill-up" with substrate (Ml), the observed effect is a leveling off of enzyme activity. This leveling off of activity can be thought of as enzyme saturation. In order to more accurately determine how much total lysozyme is in solution, the initial velocity (only the first two data points) are considered. The initial velocities are seen in Graphs 2a, 3a, 4a, and 5a. These graphs produce a linear relationship with a slope that represents the initial velocities that can then be plugged into Equation 1 and the amount of lysozyme determined.

Graph 2a also shows that there is more lysozyme activity in brown eggs than white eggs. Table 1 (see page 7) contains the ug lysozyme extracted from egg whites calculated from Equation 1. These calculations show that brown eggs consistently contain substantially more lysozyme than white eggs. This set of data also demonstrates that while increasing the incubation time for brown eggs from 15 to 30 minutes slightly increases the amount of lysozyme, this does not seem to be true for white eggs (see Table 1). The difference seen between egg whites from different colored eggshells may be due to the living conditions of the chickens. The white eggs were purchased from a local grocery store and were from a commercial egg layer. Commercial chickens are kept in very small cages and in rooms with lots of lights to stimulate egg production. The brown eggs used in this experiment were from a local farm. The chickens are kept in a large area and allowed to move freely about and only have the sunlight to stimulate egg production. Large individual laying boxes are provided as well as all the water and food they want. Instead of the actual color of the eggshell determining the amount of lysozyme present, perhaps the living conditions are more of a factor. Overly stressed commercial chickens may not produce as much lysozyme as their farm-free-range cousins may.

The effect of extraction time on the amount of lysozyme in the extraction solution also varied between brown and white eggs. As the incubation time increased for white eggs, the amount of active lysozyme found in solution decrease (see Graph 3 and 3a). It appears from this data that the ideal time for pH 8.0 extraction is 5 minutes for white eggs. The other test extraction times (15, 30, and 45 minutes) show similar amounts of active lysozyme in solution (see Table 2). "Active lysozyme" is used because the Shugar assays only can calculate the lysozyme in solution that is acting on the M. lysodeikticus cell walls. Perhaps because the extraction time was done at room temperature and other enzymes are known to degrade at room temperature, some lysozyme molecules extracted may have lost activity. Brown eggs demonstrated an ideal extraction time of 15 minutes with the pH 8.0 buffer (see Graph 4 , Graph 4a and Table 3). The difference of ideal extraction times from white to brown eggs may be due to the lysozyme in brown eggs being in some way protected from room temperature or more resistant to denaturation at room temperature. Another explanation could be a difference in the freshness of the eggs. The brown eggs used in this experiment were know to be under a month old at the end of the experiment, while the age of the white eggs could have been substantially different.

The extracted lysozyme was kept covered in the refrigerator at a temperature of 11.5 ^oC. The temperature appeared to not be cold enough to stop degradation of the lysozyme molecule. Between 8 and 30 days of being left in the refrigerator, a lot of enzyme activity was lost (see Graph 5, Graph 5a and Table 4). Although the lysozyme solutions were covered, some air still in contact with the egg white extractions. Thus lysozyme oxidation may have taken place over time, adding to enzyme deactivation. Purchased lysozyme from Sigma Chemical Co. comes in an airtight container that must be kept in the freezer (0.5 ^oC) and is in a very pure solid form. The lysozyme extracts were in solution, allowing for increased denaturation caused by the solvent.

Besides the new procedure developed for extracting lysozyme from egg whites, this experiment raises some questions as to the reasons for the differences seen between the brown and white eggs. Another consideration may be that not enough trials were completed to provide statistically accurate results. Time constraints and limited quantities of the Amberlite resin beads restricted the number of runs possible. It would be interesting to see if the results obtained could be duplicated with another couple dozen farm-fresh and grocery-store eggs.

GRAPHS

REFERENCES

1. G. Alderton, W.H. Ward, & H.L. Fevold, The Journal of Biological Chemistry, (1945), 157, 430
2. "Egg Trivia: All You Ever Need to Know", http://www.enc-online.org/trivia/.htm, last updated July 6, 1998, accessed April 5, 1999.
3. Garrett & Grisham, Biochemistry, (1995), 446-451
4. E. Li-Chan, S. Nakai, J. Sim, D.B. Bragg, & K.V. Lo, Journal of Food Science, (1986), 51, 11032
5. D. Philips, Scientific America: Biophysical Chemistry, (1957), 141
6. D. Shugar,Biochim. Biophys. Acta, (1963), 8, 376

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Last modified: July 20, 1999. JG.