Figure 2 Spectrophotometric detection of caspase activity Cleavage of the labeled colorless substrate DEVD by caspase results in the release of the chromophore pnitroaniline which can be detected by monitoring the OD at 405 nm

3.27.2.2.3 Phosphatases (semidirect assay format)

Phosphorylation by kinases and dephosphorylation by phosphatases play an important role in cell physiology and regulate metabolic and signaling events. The dephosphorylation of phosphorylated proteins or lipids can be monitored by determining the released inorganic phosphate. Phosphate is captured in a molybdate complex, which changes its absorption properties. The colored complex can be detected and quantified with a microplate reader by measuring optical density between 600 and 680 nm. The colored complex has a broad absorbance peak with a maximum at about 650 nm. The assay principle is outlined in Figure 3, and a typical protocol is described in Table 1. Usually, only four pipetting steps are needed and the duration of the assay is between 30 min and 1 h.

Classically, inorganic phosphate was determined as a complex of malachite green,10 and ammonium molybdate11 for determining the phosphatase activity of calcineurin. This method was further developed and ready-to-use reagents can be purchased (BiomolGreen from Biomol, Plymouth Meeting, PA, USA). This type of assay delivers highly reproducible results (Table 2). A disadvantage is the possible interaction of test compounds with the molybdate complex, resulting in false-positive compounds. This requires additional control experiments to exclude unspecific interactions. One of the easiest control experiments is running the assay in the presence of the potential active compounds in the absence or presence of inorganic phosphate without adding enzyme. In addition, secondary isotopic assays can further help to identify truly active compounds. In cases where inorganic phosphate is released by an enzyme reaction, this type of assay can be applied and is therefore not limited to phosphatases. Other enzyme activities that can be monitored include ATPases, pyrophosphatases, and phosphodiesterases (5'-nucleotidase coupled). Advantages for HTS compared to other methods are: (1) no radioactivity; (2) no excessive mixing; (3) high sensitivity; (4) can be miniaturized; and (5) cost effectiveness. Since the phosphate reagent is highly sensitive, the background might be increased due to free phosphate originating from lab equipment. Therefore, unused plasticware is recommended or items should be rinsed with dH2O after cleansing with detergents.

3.27.2.2.4 Protein-protein interactions (indirect format)

An assay for screening small molecule libraries was developed by Jin and co-workers12 to identify active compounds preventing the formation of the helical bundle complex of HIV gp41 molecules, which is important for virus-cell fusion

Figure 3 Phosphatase assay with molybdate. Phosphorylated substrate is enzymatically dephosphorylated and inorganic phosphate is released. In a second step, inorganic phosphate is captured in a molybdate complex. The change in absorption properties can be quantified with a microplate reader at 600-680 nm.

Measure OD at 630 nm

Figure 3 Phosphatase assay with molybdate. Phosphorylated substrate is enzymatically dephosphorylated and inorganic phosphate is released. In a second step, inorganic phosphate is captured in a molybdate complex. The change in absorption properties can be quantified with a microplate reader at 600-680 nm.

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