Methods to Characterise Enzyme Kinetics with Biological and Medicinal Substrates: The Case of Alkaline Phosphatase

Alkaline phosphatase (AP) enzymes are of broad interest in fields ranging from biochemistry and medicine to biotechnology and nanotechnology. Characterising the catalytic activity of AP is typically realised by either employing non-natural signal-generating substrates that are detectable by absorbance and fluorescence spectroscopy or by quantifying the release of inorganic phosphate by the classic malachite green assay. The latter method is often required for studying “spectroscopically silent” biomolecular substrates, but it does not enable continuous monitoring of kinetics in real-time. In recent years, newer techniques for studying AP function have been developed to circumvent this limitation, including fluorescent and colourimetric substrate-specific assays based on supramolecular chemistry, organic probes and nanomaterials, as well as other assays based on isothermal titration calorimetry, direct detection with infrared spectroscopy and mass spectrometry, and monitoring conformational change by fluorescent nanoantennas. Here, we review these strategies and comment on their strengths and weaknesses in the context of AP.