A Dye Release Assay to Quantify Enzymatic Activity of Antimicrobial Proteins

Begin with a tube containing heat-killed bacterial suspension and treat with a dye solution.

The dye molecules covalently bind to the peptidoglycan — an essential component of the bacterial cell wall, imparting a blue coloration.

Pellet the bacteria and remove the supernatant containing unbound dye molecules.

Resuspend the bacteria and introduce an antimicrobial protein solution to the tube, triggering enzymatic hydrolysis of the bacterial peptidoglycan, resulting in cell wall degradation followed by cell lysis.

This leads to the release of the dye molecules, imparting blue coloration.

Add ethanol — a protein denaturing agent, inactivating the antimicrobial proteins and terminating the enzymatic reaction.

Centrifuge the contents to pellet the cell fragments and enzymes. Transfer the dye molecule-containing supernatant to a multi-well plate.

Using a plate reader, measure the absorbance at 595 nanometers.

Compare the absorbance of the enzyme-treated sample with that of the enzyme-untreated sample to quantify the enzymatic activity of the antimicrobial protein.

In the dye release assay, the substrate is covalently linked to Remazol Brilliant Blue R dye or RBB. Begin this protocol by making a 200-millimolar RBB solution. Dissolve 1.25 grams of RBB in a freshly prepared sodium hydroxide solution.

Next, resuspend heat-killed bacterial cells at a concentration of 0.5 grams wet weight in 30 milliliters of RBB solution. For purified peptidoglycan, resuspend the peptidoglycan at a concentration of 0.3 grams wet weight and 30 milliliters of RBB solution.

Since subsequent steps are the same regardless of the substrate used, the labeling will be demonstrated for only the bacterial cells. Incubate the reaction mixture on a rotating platform for six hours at 37 degrees Celsius with gentle mixing. After six hours, transfer the reaction mixture to a 4 degrees Celsius incubator, and incubate for an additional 12 hours with gentle mixing.

When the incubation is complete, harvest the dyed substrate by centrifugation at 3,000 times g for 30 minutes. Decant the dye solution from the substrate pellet. Resuspend the pellet thoroughly in type 1 water followed by centrifugation. Wash the dye-labeled cells three to five times in this manner to remove non-covalently linked soluble dye from the substrates.

For final washes, transfer the pellet portion that will be used in the dye release assay to 1.5-milliliter microfuge tubes. The supernatant of the last wash should be clear, while the substrate remains blue. Store the dyed substrate suspended in a minimal amount of water at negative 20 degrees Celsius until ready for use.

To prepare for the dye-release assay, allow the frozen dyed substrate to return to room temperature, and wash twice with the assay buffer that was empirically determined for the enzyme. PBS is used, in this case. Calculate the volume of the substrate suspension that is needed by multiplying the 200-microliter reaction volume by the number of dye-release assays to be performed.

To prepare the substrate suspension, first add the dyed substrate to the appropriate volume of PBS. Make a one-to-one dilution and take an initial measurement of the optical density at 595 nanometers using PBS as a blank. Add incremental amounts of the dyed substrate to the suspension until an optical density of 2.0 at 595 nanometers is achieved.

In this demonstration, the dye-release assay will be used to determine the optimal incubation conditions for a protein antimicrobial. First, prepare a stock protein antimicrobial suspension by adjusting the concentration to 1 milligram per milliliter using PBS, as described in the protocol text. Then, dilute the stock protein antimicrobial suspension to a concentration of 100 nanograms per microliter.

This concentration gives a final reaction mass of 1 microgram of protein per volume added to the assay reaction. Perform the reaction assays in 0.5-milliliter microfuge tubes for the thermal range to be assessed. For each thermal condition, add 10 microliters of the stock protein to 200 microliters of prepared substrate suspension.

Incubate in a thermal cycler for eight hours with mixing by inversion once per hour. Following the incubation, arrest the reactions by adding 25 microliters of ethanol to each microfuge tube. To remove undigested insoluble substrate, centrifuge at 3,000 times g for two minutes. After that, while being careful not to disrupt the pellet of undigested substrate, transfer 150 microliters of the reaction supernatant for each reaction mixture to a 96-well flat-bottom microplate.

To quantify the enzymatic activity, use a microplate spectrophotometer to measure the absorbance of the supernatant at 595 nanometers. Increased absorbance by the soluble dye released into the supernatant from the labeled substrate is a quantitative measurement of enzymatic activity.