A Dye Release Assay to Quantify Enzymatic Activity of Antimicrobial Proteins

1. Preparation of Bacteria

NOTE: Whole-cell bacterial substrates and purified peptidoglycans are used as enzyme substrates in both the microslide diffusion assay and the dye-release assay. These substrates require preparation prior to conducting the enzyme reactions. The following protocol describes their preparation.

1. Whole Bacterial Cell Substrate
   1. Produce bacterial cell substrate by inoculating 500 ml of nutrient broth with a 2 ml overnight culture of Bacillus subtilis 168 (American Type Culture Collection; ATCC 23857). Incubate at 30 °C with shaking (125 rpm) until the culture reaches an exponential phase of growth, defined as a rapid growth phase resulting in the doubling of the bacterial culture. For the cultivation of Salmonella enterica subsp. enterica (ATCC 10708), use nutrient broth as a growth medium at 37 °C with shaking (125 rpm).
   2. Heat-kill each culture by autoclaving for 10 min at 121 °C under 3 atm of pressure.
   3. Harvest the heat-killed bacterial substrate by centrifugation for 20 min at 5,000 x g. Wash the pellet three times with Type 1 water and re-suspend in a minimal amount of water. In this study, suspend the substrates in 1,200 µl.
   4. Aliquot 300 µl of the bacterial cell substrates to 1.5 ml microfuge tubes and store at 20 °C.

2. Substrate Labeling – Remazol Brilliant Blue R Dye Labeling

NOTE: In the dye-release assay, the substrate is covalently linked to Remazol brilliant blue R dye. The following protocol describes the preparation of dyed enzyme substrates.

1. Make a 250 mM sodium hydroxide (NaOH) solution by dissolving 1 g NaOH in 99 ml of Type I water to be used for making a 200 mM Remazol brilliant blue R dye (RBB) solution.
2. Make a 200 mM RBB solution by dissolving 1.25 g RBB in 98.75 ml ± 1 ml of a fresh 250 mM NaOH solution.
3. Re-suspend heat-killed bacterial cells at a concentration of 0.5 g wet weight in 30 ml of RBB solution. For purified peptidoglycan, re-suspend the peptidoglycan at a concentration of 0.3 g wet weight in 30 ml of RBB solution.
4. Incubate the reaction mixture in an Erlenmeyer flask on a rotating platform for 6 hr at 37 °C with gentle mixing.
5. Transfer the reaction mixture to a 4 °C incubator and incubate for an additional 12 hr with gentle mixing.
6. After incubation, harvest the dyed substrate by centrifugation at 3,000 x g for 30 min. Decant the dye solution from the substrate pellet.
7. Remove non-covalently linked soluble dye from the substrates by washing the dye-labeled cells or peptidoglycan repeatedly (approximately 3-5 washes) with Type I water followed by centrifugation. With each water addition, re-suspend the pellet thoroughly.
   NOTE: When unbound, soluble dye is no longer visible in the water wash after centrifugation. The substrate should be given one additional wash. Note that the substrate will remain blue, while the supernatant of the last wash will be clear.
8. Store the dyed substrates suspended in a minimal amount of water at -20 °C for later use.

3. Quantitative Dye-release Assay

NOTE: During the dye-release assay, the hydrolysis of RBB-dyed substrate in the enzymatic reaction releases dyed products into the reaction supernatant. Colorimetric measurement of the amount of dye released indicates the amount of enzymatic activity present within the sample for a given enzyme. The quantitative method allows the comparison of different enzymes across substrates and allows for variation of environmental conditions influencing the enzymatic reaction (e.g., temperature, salinity, and pH). The following protocol describes the preparation and performance of the dye-release assay for quantitatively detecting the enzymatic activity of protein antimicrobials.

1. Preparation for the Dye-release Assay
   1. Allow the frozen dyed substrate to return to room temperature and wash twice with assay buffer (phosphate-buffered saline, PBS), which is empirically determined for the given enzyme.
   2. Calculate the volume of substrate suspension that is needed by multiplying the 200 µl reaction volume by the number of dye-release assays to be performed.
   3. Prepare the substrate suspension by adding dyed substrate to the volume of assay buffer, determined in 4.1.2, until an optical density (OD) of 2.0 is achieved at 595 nm using a spectrophotometer. To remain within the functional limitations of the spectrophotometer, measure the 2.0 OD₅₉₅ as 1.0 for a 1:1 dilution of the concentrated solution. Use assay buffer as a blank.
      NOTE: The substrate turbidity for the reaction can be raised beyond 2.0 to match the activity levels of highly efficient enzymes.
   4. Suspend the protein antimicrobial to be evaluated in assay buffer at an estimated concentration of 1 mg/ml.
   5. Using the microplate assay of a Bicinchoninic acid (BCA) Protein Assay Kit according to the manufacturer's protocol, determine the actual concentration of the protein sample to be assayed. Adjust the concentration of the stock suspension to 1 mg/ml using assay buffer.
2. Using the Dye-release Assay to Determine the Optimal Incubation Conditions for a Protein Antimicrobial
   1. Dilute the stock protein antimicrobial suspension to 100 ng/µl. This concentration gives a final reaction mass of 1 µg of protein per volume (10 µl) added to the assay reaction.
   2. Determine the thermal range to be assessed for the bacteriolytic protein. The thermal range in these assays included 5 °C, 15 °C, 25 °C, 35 °C, 45 °C, 55 °C, and 65 °C.
   3. Perform the reaction assays in 0.5 ml microfuge tubes. For each thermal condition, add 10 µl of the stock protein to 200 µl of prepared substrate suspension.
   4. Incubate in a thermal cycler for 8 hr with mixing by inversion once per hour.
   5. Following incubation, arrest the reactions by adding 25 µl of ethanol.
   6. Remove undigested, insoluble substrate by centrifugation at 3,000 x g for 2 min, and transfer 150 µl of the reaction supernatant for each reaction mixture to a 96-well flat-bottom microplate, taking care not to disrupt the pellet of undigested substrate.
   7. Measure the enzymatic activity of the protein antimicrobial for the given substrate by determining the amount of soluble RBB dye dissociated from the substrate after enzymatic hydrolysis. To quantify the enzymatic activity, measure the absorbance of the supernatant at 595 nm using a microplate spectrophotometer. Increased absorbance by the soluble dye released into the supernatant from the labeled substrate is a quantitative measurement of enzymatic activity.       
      NOTE: The change in absorbance is represented by activity units (AU), where 1 AU results in a 0.01 increase in the optical density of the dye-release reaction supernatant at 595 nm. A blank reaction, incubated with all reaction components except the enzyme, is used to subtract any dye that is released from the RBB-labeled substrate due to the incubation. The optimal enzymatic temperature provides the peak activity unit measurement.