Analytical Biochemistry 623 (2021) 114170 Available online 15 March 2021 0003-2697/© 2021 Elsevier Inc. All rights reserved. Technical note An antimony-phosphomolybdate microassay of ATPase activity through the detection of inorganic phosphate Jordan L. Pederick , John B. Bruning * Institute for Photonics and Advanced Sensing (IPAS), School of Biological Sciences, The University of Adelaide, Adelaide, South Australia, Australia A R T I C L E INFO Keywords: ATPase Inorganic phosphate Molybdenum blue Phosphate assay Enzyme assay ABSTRACT Colorimetric methods are convenient for the determination of inorganic phosphate. However, the acidic con- ditions required can complicate measurement of ATPase through non-enzymatic ATP hydrolysis. Here we present an optimized antimony-phosphomolybdate microassay for the simple and rapid detection of ATPase activity, with micromolar sensitivity. The low acidity of the color reagent results in no interference for samples containing up to 0.5–5 mM ATP, dependent on the sample volume. The assay is compatible with common assay conditions and was similar in accuracy to an established continuous method. The simplicity of this method makes it ideal for medium to high throughput applications. 1. Introduction The colorimetric determination of inorganic phosphate (P i ) is a common approach for measuring ATPase activity. Ideally, these methods are simple and rapid while maintaining sensitive and accurate detection. Currently, these are based on the formation of a complex between P i and molybdic acid, being iterations of the malachite green and molybdenum blue assays [1,2]. The former involves the formation of a complex between the dye malachite green and phosphomolybdate, producing a species with an absorbance maximum in the range 600–700 nm. The method utilizes a single color reagent (CR) and detects P i at sub micromolar concentrations (ε ≈ 80 000 M 1 cm 1 ), with color devel- opment generally complete after 10–30 min. The main limitation of this method for measuring ATPase activity is the non-enzymatic hydrolysis of ATP due to the requirement of a strong acid to stabilize the dye complex. This results in the release of P i during the color development step which can interfere with determination of enzymatically released P i when ATP is present at low micromolar concentrations [3]. Therefore, accurate determination of ATPase activity using this method can require dilution of samples coupled with precise timing of quenching and the use of blank wells for subtraction. Alternatively, a chelating agent such as citric acid has been commonly used to quench color development and provide a stable signal, however this introduces an additional step and reagent to the assay. Therefore, alternative colorimetric methods that overcome this limitation are of interest. The molybdenum blue assay involves the reduction of phosphomo- lybdate to form a species with maximum absorbance between 800 and 900 nm allowing detection of P i at micromolar concentrations (ε ≈ 20 000 M 1 cm 1 ). This method and its various derivatives have generally been used for characterizing membrane ATPases as less acidic condi- tions are required, allowing higher ATP content of samples. Addition- ally, the detergent sodium dodecyl sulfate (SDS) is compatible with the method, allowing determination of P i in samples with high protein content. In 2013, Bartolommei et al. described a variation of the mo- lybdenum blue assay for detecting ATPase activity through the forma- tion of an antimony-phosphomolybdate complex [4]. This method combines several advantageous properties, including rapid color development, a single stable and low-cost CR, and a low rate of ATP hydrolysis. These characteristics are ideal for medium and high throughput applications, however this cuvette-based assay has yet to be exploited in a miniaturized format. Previously, we adapted this method for assaying the activity of the ATP-grasp enzyme D-alanine-D-alanine ligase from Thermus thermophilus and found it to be robust for kinetic analysis [5]. Here we present an optimized antimony-phosphomolybdate microassay based on this previous work that allows simple and rapid detection of ATPase activity. Abbreviations: CR, color reagent; KH 2 PO 4 , potassium phosphate; SDS, sodium dodecyl sulfate; Ddl, D-alanine-D-alanine ligase; DCS, D-cycloserine. * Corresponding author. E-mail address: john.bruning@adelaide.edu.au (J.B. Bruning). Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio https://doi.org/10.1016/j.ab.2021.114170 Received 20 January 2021; Received in revised form 28 February 2021; Accepted 6 March 2021