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