A Universal, Fully Automated High Throughput Screening Assay for Pyrophosphate and Phosphate Release from Enzymatic Reactions

Abstract

The malachite green assay is often used for measuring the presence of inorganic mono-phosphate concentrations. Some studies have adapted this assay for use in monitoring enzymatic reactions and have suggested its potential use in high throughput screening (HTS). With the increasing availability of laboratory automation, some studies are starting to explore the possibility of conducting limited, semi-automated versions of the assay. Here we report the optimization and complete adaptation of the malachite green assay to a fully automated, HTS platform that can be performed unattended with standard, commercially available, automated liquid-handling systems. The assay is universal for the majority of enzymes that release phosphate or pyrophosphate. Moreover, the assay is fully scalable from smaller drug screening efforts ( approximately 20,000 wells per day) to ultra-high throughput environments ( approximately 200,000 wells per day). The assay uses cost-effective, commercially available reagents, and can be used to perform automated IC50 value and kinetic parameter determination. Finally, we demonstrate the utility of the assay via the initial, primary screening of 100,080 compounds against two target enzymes from Bacillus anthracis, O-succinylbenzoyl-CoA synthetase and nicotinate mononucleotide adenylyltransferase.

Comparative analysis and validation of the malachite green assay for the high throughput biochemical characterization of terpene synthases

“…Enzymatic reactions were quenched by addition of 12 μL of the malachite green development solution and incubated for 15 min prior readings at 623 nm on a Varioskan Flash plate reader. The malachite green development solution was prepared fresh before each experiment by mixing 10 mL of malachite green dye stock solution with 2.5 mL of 7.5% ammonium molybdate and 0.2 mL 11% Tween 20 [6]. According to our experience the development solution can be used for up to 5 hours after mixing.…”

“…According to our experience the development solution can be used for up to 5 hours after mixing. The malachite green dye stock solution was prepared according to Pegan et al [6]. In short, 300 mL of 18 M sulfuric acid stock was mixed with 1.5 L of water and then cooled down to room temperature.…”

Continuous Enzyme-Coupled Assay of Phosphate- or Pyrophosphate-Releasing Enzymes

“…In addition, chemosensors for phosphates were recently designed, synthesized, and tested (1,2). Using traditional or chemosensor-assisted chemical methods, phosphates can be detected using sensitive, simple, and cost-effective procedures, some of which can also be miniaturized (3) and fully automated (4). Nevertheless, these procedures are discontinuous, rendering rather cumbersome the kinetic evaluation of reactions involving phosphates.…”

Differences in the purification and solution properties of PurC gene products from Streptococcus pneumoniae and Bacillus anthracis

“…Reaction solution (70 lL) was quenched with malachite green (17.5 lL; BioAssay Systems) in 384 well format [14]. Negative controls lacked PurC.…”