Good-Practice Non-Radioactive Assays of Inorganic Pyrophosphatase Activities

Abstract

Inorganic pyrophosphatase (PPase) is a ubiquitous enzyme that converts pyrophosphate (PP_i) to phosphate and, in this way, controls numerous biosynthetic reactions that produce PP_i as a byproduct. PPase activity is generally assayed by measuring the product of the hydrolysis reaction, phosphate. This reaction is reversible, allowing PP_i synthesis measurements and making PPase an excellent model enzyme for the study of phosphoanhydride bond formation. Here we summarize our long-time experience in measuring PPase activity and overview three types of the assay that are found most useful for (a) low-substrate continuous monitoring of PP_i hydrolysis, (b) continuous and fixed-time measurements of PP_i synthesis, and (c) high-throughput procedure for screening purposes. The assays are based on the color reactions between phosphomolybdic acid and triphenylmethane dyes or use a coupled ATP sulfurylase/luciferase enzyme assay. We also provide procedures to estimate initial velocity from the product formation curve and calculate the assay medium's composition, whose components are involved in multiple equilibria.

Keywords: ATP sulfurylase; initial velocity; luciferase; malachite green; methyl green; phosphate assay; pyrophosphate assay; pyrophosphate complexes.

Figure A1

Time-course of product accumulation in the enzyme-catalyzed reaction (solid line). The dashed straight line is the zero-time tangent to the solid curve, with the slope equal to the initial velocity.

Figure A2

The dependence of ∆P in Figure A1 on P_inf at seven fixed values of P (shown on one panel and kept same on the other panels) and six values of K_m (shown on the panels) for an enzyme-catalyzed reaction. For simplicity, the values of ∆P, P_inf, P and K_m are shown in terms of the same arbitrary unit, for instance, recorder paper division. These graphs were created for an enzyme that obeys simple Michaelis–Menten kinetics with the product to substrate ratio of one. To use these graphs for PPase, which produces two P_i molecules from one PP_i molecule, one must divide by two the measured values of P and P_inf if they are expressed in terms of P_i. Note that axis scaling is different on the two bottom panels.

Figure 1

The phosphate analyzer is used to assay PPase activity in a continuous way. (A) Flow diagram for the phosphate analyzer in standard mode; (B) Tubing connections on the pump in the high-sensitivity mode; (C) tubing connections in the low dead-time mode. Numbers on the pump refer to flow rate in mL/min (before the slash) and tubing diameter in mm (panel A) or flow rate in mL/min (panels B and C). (D) Actual P_i accumulation recordings in setup A with photometer sensitivity of 1 absorbance unit per recorder scale. The calibration data shown at the beginning of the recording was obtained by adding 0–200 µM P_i to the reaction buffer. (E) Actual P_i accumulation recordings in setup B with photometer sensitivity of 0.1 absorbance unit per recorder scale. The assay mixture of 40 mL volume contained 140 µM PP_i, 5 mM MgCl₂, 50 mM MOPS–KOH, pH 7.2, and 0.03 nM Streptococcus gordonii PPase with a specific activity of 480 s⁻¹. (F) Actual recordings of P_i accumulation in the setup C for rat liver PPase in the presence (a) or absence (b) of slow-binding inhibitor (10 mM fluoride). The arrow marks the moment of enzyme addition. Part of the figure was taken with permission from references [13] (panels A and D) and [14] (panels C and F).

Figure 2

PPase assay using the malachite green procedure. Two bottom rows show a duplicate series of phosphate dilutions from 0 µM (left) to 10 µM (right). Two top rows show typical results of a duplicate screening test of a library of potential inhibitors of Escherichia coli PPase. The yellow color indicates strong inhibition, dark green color—no inhibition.

Figure 3

Schematics of the assays to measure PPase-catalyzed PP_i synthesis. (A) A continuous assay of the medium PP_i synthesis; (B) fixed-time assay of the medium PP_i synthesis; (C) determination of enzyme-bound PP_i. The assayed PPase is added to all far-left tubes. Three other major components are shown as colored spots. The principal analytes transferred between the tubes are indicated above the arrows. The blue star refers to the luminescence signal. (D) Actual PP_i accumulation recordings in the assay version A for baker’s yeast PPase in the presence of slow-binding inhibitor (fluoride; its concentrations in mM are indicated on the curves). Panel D was taken with permission from reference [34].

Scheme 1

Complex formation between PP_i, H⁺, Mg²⁺, K⁺ and Na⁺ ions under physiological conditions. The number above or beside arrows refers to the minus logarithm of the respective dissociation constant [41].

Scheme 2

A simplified description of complexation between PP_i and Mg²⁺ at fixed pH and alkali metal ion concentrations.

Scheme 3

Complex formation between P_i, H⁺, Mg²⁺, K⁺, and Na⁺ ions under physiological conditions. The number above or beside arrows refers to the minus logarithm of the respective dissociation constant [42,43,44,45].