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1. Reagents for the assay preparation
- Prepare the arsenite-citrate solution.
- Weigh 5 g of sodium arsenite and 5 g of trisodium citrate dihydrate.
CAUTION: Sodium arsenite is toxic; thus use proper protective equipment and handle with special care. As a precaution, do not handle before all necessary safety precautions have been read and understood. Handle only in a fume hood in order not to inhale dust/vapors of the compound or its solution(s). If inhaled, move to fresh air and obtain medical attention. Wear appropriate chemical safety goggles, protective gloves, and clothing to avoid ingestion and eye/skin contact. If swallowed, call immediately a poison center or doctor/physician. If it gets on the skin or in the eye(s), wash with plenty of water and obtain medical attention. - Dissolve into 100 mL of water.
- Add 5 mL of glacial acetic acid, mix, and add water to 250 mL.
- Store at room temperature, protected from light.
NOTE: The solution is stable for more than a year.
- Prepare solution A and solution B.
- For solution A, add 10 mL of ice-cold 0.5 M HCl (hydrochloric acid) to 0.3 g of ascorbic acid. Dissolve the ascorbic acid by vortexing.
- For solution B, add 1 mL of ice-cold water to 70 mg of ammonium heptamolybdate tetrahydrate and vortex to dissolve.
NOTE: Store both solutions on ice until use. For the consistency of the assay result, both solutions can be stored on ice for a maximum of one week.
- Prepare the phosphate (Pi) standard with the concentration of 0 µM, 62.5 µM, 250 µM, and 500 µM for calibration.
- Add 0 µL, 25 µL, 50 µL, and 100 µL of 5 mM Na2HPO4 (disodium phosphate) dihydrate to four microtubes containing 370 µL of the reaction mixture.
- Top up to 1 mL with water.
2. Activity assay for one 96-well plate
NOTE: See Figure 1 for the schematic workflow of the assay.
- Add 1 mL of solution B to 10 mL of solution A, mix by vortexing, and store the solution on ice.
NOTE: This solution should be transparent and yellow. Keep solution A + B on ice for at least 30 min prior to use. However, use the solution within 3 h as it will go bad after long-term storage. - Add 40 µL of 0 µM, 62.5 µM, 250 µM and 500 µM Pi standard to the tube strips in triplicate using a multichannel pipette.
NOTE: The reaction mixture with no Pi added will be used as a blank. - Add 25 µL of compound solution to the tube strips using a multichannel pipette.
NOTE: Each compound has three different concentrations in triplicate which is enough for initial estimation of the half maximal inhibitory concentration (IC50). For a more accurate IC50 determination, eight different compound concentrations can be used. For the uninhibited enzyme the compound solution is replaced with equal amount of water. As positive controls 2.5 µM, 25 µM, and 250 µM of imidodiphosphate (IDP) sodium salt were used. - Add 15 µL of mPPase (membrane-bound pyrophosphatase) solution mixture to the tube strips (except for the tubes containing Pi standard) using a multichannel pipette.
- Seal the tube strips with an adhesive sealing sheet. Cut the sealing sheet to separate each tube strip.
- Pre-incubate the samples for 5 min at 71 °C. Place the samples on the heating block with 20 s interval between each strip in order to minimize the time consumption during the subsequent steps.
- For each strip, open the adhesive sealing. Add 10 µL of 2 mM sodium pyrophosphate dibasic using a multichannel pipette and mix by pipetting up and down for 5×. Seal the tube strip again using the same sealing.
NOTE: This step might initially be difficult to accomplish in 20 s; however, it will become easier after some assays. - Incubate at 71 °C for 5 min.
- Place the samples on the cooling apparatus with 20 s interval between each strip. Let them cool for 10 min but centrifuge each strip briefly after 5 min of cooling, to decant water drops under the sealing sheet, then put it back to the cooling apparatus and remove the sealing.
NOTE: The cooling apparatus can simply be made by placing a 96-well PCR (polymerase chain reaction) plate on a polystyrene Petri dish (size 150 mm × 15 mm) filled with water and frozen for at least 1 h. The apparatus should be taken out from the freezer about 5 min prior to the beginning of the assay. Do not take out the cooling apparatus right before sample cooling as it will freeze the reaction mixture and hinder color development. - After 10 min of cooling, add 60 µL of solution A + B, mix by pipetting up and down for 5×, and keep the tube strips on the cooling apparatus for 10 min.
- Add 90 µL of the arsenite-citrate solution and keep at room temperature for at least 30 min to produce a stable blue color.
CAUTION: Due to its toxicity, all solutions containing sodium arsenite should be handled with extra care at all time. Thus, the addition of arsenite-citrate solution should be done in a fume hood. - Dispense 180 µL of each reaction mixture into a clear 96-well polystyrene microplate.
- Measure the absorbance of each well at 860 nm using a microplate spectrophotometer.
