Thin Layer Chromatography Based Enzyme Activity Assay: A Technique to Determine In Vitro Pyrophosphokinase Activity Using Thin Layer Chromatography

Pyrophosphokinase catalyzes diphosphate or pyrophosphate transfer from nucleotide precursor, like adenosine triphosphate, ATP, to substrate like guanosine diphosphate, GDP. This forms guanosine tetraphosphate - an important signaling molecule.

To determine pyrophosphokinase activity, prepare a reaction mixture comprising GDPs and radiolabeled ATPs in a suitable buffer supplemented with magnesium chloride. Add the desired amount of pyrophosphokinase to the reaction mixture and incubate.

Over time, in the presence of magnesium ions, pyrophosphokinase starts transferring the radiolabeled pyrophosphate moiety from ATP to GDP, producing radioactive guanosine tetraphosphate.

Now, take a thin layer chromatography or TLC, plate precoated with polyethylenimine-cellulose - a cationic polymer-based stationary phase. Spot aliquots of the reaction mixture analytes near the TLC plate base at different time points to stop the reaction and evaluate progress.

Immerse the plate in a glass chamber containing monobasic potassium phosphate - a mobile phase solvent. As the solvent front moves upward, negatively charged analytes begin to migrate based on their affinities with the positively charged stationary phase.

The differences in the negative charges and weight of differently phosphorylated analytes determine their relative migration on the TLC plate, allowing them to immobilize as distinct bands.

As the solvent front reaches the desired distance, remove the plate and air-dry. Measure the radioactive signal intensity, corresponding to radioactively labeled ATP depletion and guanosine tetraphosphate accumulation to analyze the pyrophosphokinase activity.

Prior to performing the reaction, prepare PEI-cellulose thin-layer layer chromatography plates by washing them in deionized water. Place the plates in a glass chamber with double distilled water, to a depth of approximately 0.5 centimeters. After allowing the water to migrate to the top of the plate, bring the plates out of the glass chamber, and leave on a benchtop rack to dry overnight.

Mark the dry plates 2 centimeters from one edge with a soft pencil, to indicate where the samples will be applied for TLC. For 2 microliter samples, apply samples no less than 1 centimeter apart. When planning experiments, always leave one spot on each plate unused, to serve as a blank lane for sample quantification.

To perform the enzyme activity assay, prepare individual reactions using γ-³²P-ATP, as described in the text protocol. Add the RSH after the other components have been mixed, as the addition of RSH to the nucleotide-containing mix initiates the enzymatic activity assay.

To control for ATP hydrolysis from contaminating nuclease activity, assemble a 10-microliter reaction containing no protein, and incubate it in parallel. Spot 2 microliter samples at t = 0, and at the end of the experiment to ensure that ATP was not hydrolyzed in the absence of protein.

Immediately upon addition of RSH, remove 2 microliters, and spot it onto the labeled PEI-cellulose plate as the t = 0-minute sample.

Incubate the reaction at 37 degrees Celsius, removing 2 microliter aliquots at desired time points. After all aliquots have been collected, perform thin-layer chromatography by filling the chromatography chamber with 1.5 molar monobasic potassium phosphate to a depth of 0.5 centimeters. Immerse the bottom edge of the plate in solvent, and allow the solvent to migrate to the top of the plate over approximately 90 minutes.

Remove the plate from the chromatography tank, and place it on a benchtop drying rack to air-dry overnight. After the plate is dry, wrap the plate in plastic film to avoid transfer of radioactive material to the imaging cassette, and analyze by autoradiography.

Expose the PEI-cellulose plate containing separated reactions to a phosphorimager cassette for four hours at room temperature. Following exposure, image the cassette on a phosphorimager.

Using imaging software with a graphical user interface, draw regions of interest, or ROIs, by first selecting "Draw rectangle." Then, use the mouse to draw rectangular ROIs around one entire lane, and the ATP and guanosine tetraphosphate spots contained within that lane.

Use the "Select, Copy, and Paste" commands to draw identical ROIs within the other lanes, to ensure that the ROIs are measuring signals within identical areas in each lane. Include ROIs from an unused lane to be used as blanks.

Using the "Analyze | Tools | ROI Manager | Add" commands of the imaging software, select all of the ROIs drawn on the PEI-cellulose plate. Now, use the "Analyze | Set Measurements | Measure" commands to quantify the signal intensity within each ROI, and export the measurements as a spreadsheet. In the spreadsheet, subtract blank ROI values from experimental signals.