Changes In Hemolymph During the Antibacterial
Response of M. sexta
Protocol: Determination of Lysozyme Activity in
Manduca sexta Hemolymph
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Lysozyme is an enzyme that attacks bacterial cell walls.
It degrades the cell wall by cleaving the sugar backbone of
the peptidoglycan component. Specifically, lysozyme adds
water to (hydrolyzes) the glycosidic bond between
N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG).
Lysozyme is a common constituent of biological tissues and
secretions; it has been found in egg whites, tears, sweat,
the digestive tract of ruminants and the hemolymph of
Bacterial cell walls are of two types: Gram negative and
Gram positive. Gram positive bacteria have a cell
wall composed of a thick layer of peptidoglycan overlaid by
a thinner layer of techoic acid. In contrast, Gram
negative bacteria have a thinner layer of
peptidoglycan which is enclosed in a second lipid bilayer.
(The 'Gram' designation comes from whether or not the
specific bacteria is stained by a reaction series developed
by a Danish physician, Hans Christian Gram.) Lysozyme is
most effective against Gram positive bacteria since the
peptidoglycan layer is relatively accessible to the enzyme;
lysozyme is effective against Gram negative bacteria only
after the outer membrane has been compromised.
An enzymatic assay is used to determine the amount of
enzymatic activity present in a given sample. In this
experiment, we will determine the lysozyme activity present
in hemolymph samples from two M. sexta larvae (those
that you used in the protein determination assay last week).
The lysozyme assay that we will perform uses Micrococcus
lysodeikticus bacterial cells as a substrate for the
lysozyme. A suspension of the M. lysodeikticus cells
will be mixed with a small amount of hemolymph. The lysozyme
present in the hemolymph will degrade the cell walls. As the
enzyme acts on the suspension, the turbidity of the
suspension, and therefore, the absorbance of the sample,
will decrease. The rate of decrease in the turbidity
will be a relative measure of the amount of lysozyme present
in the sample; the more lysozyme present, the faster the
reaction occurs and the faster the absorbance decreases.
(per lab group)
- mg Micrococcus lysodeikticus cells in beaker
catalog number M3770)
- small bottle of assay buffer (0.1 M KPO4
pH 6.4, 0.02% NaN3)
- cuvettes (2)
- water bottle
- 25 ml graduated cylinder
- stir plate
- rubber stopper
- beaker for waste
- micropipettors for measuring up to 1 ml and 0.2
- pipette tips for above
- stir bar (small)
- ice bucket
- lab tissues
- stop watch
The only hazardous chemical present in this experiment is
the NaN3 (sodium azide) in the assay buffer. This
chemical disrupts electron transport in mitochondria. It may
be fatal if swallowed, inhaled or absorbed through the skin.
It is a neurotoxin and suspected mutagen. Please wear
gloves, eye protection and lab coats. Wash your hands before
leaving the lab.
1. Obtain a beaker that contains 9 mg of M.
lysodeikticus cells. Add the stir bar. Add 25 ml of
assay buffer. Place the beaker on the rubber stopper on top
of the stir plate (the stopper acts as an insulator so that
the cell suspension does not absorb heat). Set the stirring
speed to a slow rate and allow the suspension to stir
for at least 5 min.
2. Turn on the spectrophotometer and allow it to warm up.
Adjust the wavelength to 570 nm.
3. Obtain your hemolymph samples from untreated and
bacteria-treated larvae (either freshly collected or stored
frozen; if frozen, place the tubes on ice to thaw
4. Read through the following assay steps 5-9. Make sure
that you understand each step. Fill in the time column for
Table 1 in the 'Results' section.
Coordinate team member activities for the assay (suggested
jobs: timing, recording, mixing, plotting the data). You may
want to do a practice round that you do mot record. Simply
use the hemolymph sample that has the largest volume to run
the assay for practice.
5. Pipette 1940 µl (2 x 970 µl) of the cell
suspension into a cuvette. Place the cuvette in the
spectrophotometer and record the reading as the beginning
absorbance for the assay. Remember that the absorbance is
going to decrease; a reading of >0.7 is best. Allow one
minute to pass and be sure that the reading remains
6. Add 60 µl of assay buffer to the cuvette. Mix by
moving the pipette tip quickly back and forth for a fixed
number of strokes (20 is good). The moment that you close
the door of the spectrophotometer, begin timing. Read the
absorbance at 1 min. intervals for 3 min. Record your data
in tabular form in the 'Results' section, Table
1. This step ensures that the assay buffer alone does
not contain lysozyme activity and the reading should not
change over the 3 minute period.
7. Remove the cuvette, empty the contents into the waste
beaker and rinse the cuvette with water. Pipette another
1940 µl of the cell suspension into the cuvette. Place
the cuvette back in the spectrophotometer and observe the
absorbance for about 15 seconds. It should be steady. Make
note of the beginning absorbance.
8. Pipette 60 µl the untreated hemolymph sample into
the cuvette. Quickly mix as in step 6. Close the
spectrophotometer and immediately begin timing. Take a
reading at 15 seconds and then at 15 second intervals for 3
9. Repeat step 8 to perform a duplicate assay for the
hemolymph sample. After looking at the data, use your
judgment about performing a third assay on that sample. The
best lab protocols perform triplicate assays but in the
interest of time and conserving hemolymph you may decide
whether a third assay is appropriate. What criteria will you
use to make this decision?
10. Repeat steps 8 and 9 for the bacteria-treated
11. While collecting the data, plot the data on a graph,
absorbance (y-axis) vs. time in minutes (x-axis).
12. The slope of the line on the graph is proportional to
the rate of the reaction. Your line may be curved either at
the beginning and/or the end of the time period. Select a
range over which the line is linear. Determine the slope of
the line (Æy/Æx) for this time period. This will give a rate
in change of absorbance units per minute. You should use the
same time period for all of your samples. Ideally, the class
should use the same time period so that data can be compared
among laboratory groups.
13. Convert this rate to enzyme units (EU) using the
conversion factor one enzyme unit = 0.01 absorbance units
14. A defined standard unit is EU/ml of hemolymph sample.
Convert your enzyme unit value above to EU/ml of hemolymph.
Be careful of units!!!
15. Record your results in Table
Data Sheets for this
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