Biology Section______ Name__________________________________
Dr. Wexler
Lysozyme Assay of Human Secretions
Date_______


Introduction:


Bacteria are single-celled prokaryotic microorganisms. They look like this:




They are protected by cells walls that contain a tough layer of polysaccharide called peptidoglycan.

The cell wall looks like this:





The peptidoglycan monomer subunit looks like this:







When assembled, the peptidoglycan layer of the cell wall looks like this:



The strength of the peptidoglycan is due to the peptide cross-bridges between adjacent NAM-NAG chains.


The main function of peptidoglycan is to preserve cell integrity by withstanding the turgor pressure inside the cell.

If peptidoglycan is disrupted, the cell will burst and die.





Lysozyme is an enzyme that is capable of breaking the covalent bonds connecting peptidoglycan monomers through a hydrolysis reaction as illustrated below:





The lysozyme primary structure is as follows:



The lysozyme secondary structure is as follows:



The lysozyme tertiary structure is as follows:




Notice the cleft on the right side. This is the enzyme active site. This is where the peptidoglycan substrate binds to the enzyme and is acted upon chemically.


Lysozyme's active site is a long, deep cleft in the protein surface. This cleft binds the polysaccharide peptidoglycan. However, it isn't a perfect fit. One of the covalent bonds between monomers must be distorted to fit inside - this increases the strain on the bond, weakening it and lowering the activation energy for hydrolysis.










Purpose:

To assay for the presence of lysozyme in human saliva or tears.


Procedure:

  1. Pipet 1 ml Micrococcus lysodeicticus suspension into each of 3 test tubes.

  2. Label the test tubes “S” for saliva, “T” for tears, or “N” for negative control. Also include your group initials.

  3. Place a dry cotton swab into the tube labeled “N”.

  4. Place a cotton swab moistened with saliva into the tube labeled “S”.

  5. Place a cotton swab moistened with tears into the tube labeled “T”.

  6. Incubate the tubes at 37°C (98°F).

  7. Examine after 30 minutes.


Results:


Sample

Negative Control

Saliva

Tears

Turbidity*

4



* scale 0-4, where 0 is clear and 4 is the turbidity of the negative control.



Discussion:

  1. Were you able to detect lysozyme activity in saliva?

  2. Were you able to detect lysozyme activity in tears?

  3. Which had the most lysozyme activity?

  4. Speculate why saliva and tears contain lysozyme:




  5. Speculate why one secretion appears to have more lysozyme than the other: