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Changes In Hemolymph During the Antibacterial Response of M. sexta

 

Protocol: Determination of Lysozyme Activity in Manduca sexta Hemolymph

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Introduction

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 Lepidoptera.

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.

 

Materials

(per lab group)

  • spectrophotometer
  • mg Micrococcus lysodeikticus cells in beaker (Sigma, 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 ml
  • pipette tips for above
  • stir bar (small)
  • ice bucket
  • lab tissues
  • stop watch

 

Hazards:

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.

 

Method:

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 slowly).

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 approximately constant.

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 minutes.

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 hemolymph sample.

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 (y/x) 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 per min.

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 2.

 

Results:

Data Sheets for this protocol.

 

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March 1999