Our protocol works as a tool to find compounds that inhibits de-activity of membrane bound pyrophosphatase. And this inhibitors may be used in the treatment of several parasitic diseases. This protocol is cheap and simple producing stable color for a long time without showing any interference in the presence of high phospholipid concentration required for enzyme reactivation.
Prepare 10 milliliters of the reactivation buffer solution, containing 20 millimolar M-E-S at PH 6.5, 3.5%volume by volume glycerol, two millimolar D-T-T, and 0.05%D-D-M. Prepare 10 milliliters of the reaction mixer, containing 200 millimolar tris hydrochloride at PH eight. Eight millimolar magnesium chloride, 333 millimolar potassium chloride, and 67 millimolar sodium chloride.
To prepare 30 milligrams per milliliter liposomes for enzyme reactivation. First, add 10 milliliters of 20 millimolar of tris hydrochloride at PH eight, with one millimolar D-T-T to 0.3 grams of L alpha phosphatidylcholine from soy bean. Put the liposome on ice and sonicate with one second post-intervals for one minute.
Pause for one minute, and repeat until the solution becomes transparent yellow. Aliquot the liposomes in 100 microliters, freeze the liquid nitrogen and store at negative 80 degrees Celsius until used. To reactivate the enzyme, thaw the liposome solution at room temperature.
Mix 40 microliters of the liposome solution with 22.5 microliters of 20%D-D-M. Keep the mixture at 55 degrees Celsius for 15 minutes. And allow it to cool to room temperature.
Then, add 36.5 microliters of the reactivation buffer solution.Mix. And add one microliter of concentrated protein to make a total concentration of 0.13 milligrams per milliliter. Next, transfer 20 microliters of the reactivated enzyme to 1480 microliters of the reaction mixture.
Mix gently and use immediately. First, dissolve the compounds in D-M-S-O to make stock solutions of 50 millimolar in 200 microliters. For soluble compounds dilute the stock solution with water to one milliliter in micro tubes, to give two, 10, and 100 micromolar.
For each dilution, vortex the compound solution for proper mixing. After that, check for compound divergation, and using a multichannel pipet, dispense 75 microliters of the reaction mixture into each well in a 96-well plate. Add 75 microliters of each compound and water as control in triplicate, and mix by pipetting up and down five times.
Measure each well at 300 volts using a microplate nephelometer. To prepare the arsenite citrate solution, weight five grams of sodium arsenite and five grams of trisodium citrate dihydrate, dissolve both compounds into 100 milliliters of water. Then, add five milliliters of glacial acetic acid.
Mix, and add water to 250 milliliters, store at room temperature protected from light. Next, prepare solution A by adding 10 milliliters of ice cold 0.5 molar hydrochloric acid to 0.3 grams of ascorbic acid Dissolve the ascorbic acid by vortexing. Prepare solution B by adding one milliliter of ice cold water to 70 milligrams of ammonium tetrathiomolybdate tetrahydrat, and vortex to dissolve.
Store solution A and B on ice. To prepare the phosphate standard with a concentration of zero, 62.5, 250 and 500 micromolar for calibration. Add zero microliters, 12.5 microliters, 50 microliters, and 100 microliters of 5 millimolar disodium phosphate dihydrate to four micro tubes containing 370 microliters of the reaction mixture.
Top up to one milliliter with water. Now add one milliliter of solution B to 10 milliliters of solution A, mix by hand and store the solution on ice. Using a multichannel pipet, add 40 microliters of zero, 62.5, 250, and 500 micromolar phosphate standard to the tube scripts in triplicate.
Then add 25 microliters of compound solution and 15 microliters of M-P-P-A solution mixture of each tube. Except the tubes containing phosphate standard. Seal the tube strips with an adhesive sealing sheet.
Cut the sealing sheet to separate each tube strip. Next, pre incubate the sample for five minutes at 71 degrees Celsius. Place the sample on the heating block with 20 second intervals between each strip.
For each strip, open the adhesive sealing. Using a multichannel pipet, add 100 microliters of two molar sodium pyrophosphate dibasic. And mix by pipetting up and down for five times.
Seal the tube strip again using the same sealing. Incubate again at 71 degrees Celsius for five minutes. After that, place the samples on the cooling apparatus with 20 second interval between each strip.
Let them cool for five minutes and then centrifuge each strip briefly to decant the water drops under the sealing sheet. Put the strip back to the cooling apparatus, remove the sealing and let them cool for another five minutes. Then add 60 microliters of solution A and B, mix by pipetting up and down for five times, and keep the tube strips on the cooling apparatus for 10 more minutes.
In a fume hood, add 90 microliters of the arsenite citrate solution and keep at room temperature for at least 30 minutes. To produce a stable blue color. Dispense 180 microliters of each reaction mixture into a clear 96-well polystyrene microplate.
Use a microplate spectrophotometer to measure the absorbance of each well at 860 nanometers. In this protocol, eight compounds were tested together with I-D-P, a common inhibitor of pyro phosphatase as a positive control. After the addition of solution A and B, and arsenite citrate, a blue color in 30 minutes of incubation at room temperature.
All three concentrations of non inhibiting compounds, displayed the same blue color intensity. As well as E-1 to E-3 without any inhibitor. The plot of enzymatic activity against the concentration of each tested compound, shows that the compound with inhibition activity formed a nonlinear curve fitting.
The plot of I-D-P as a positive control, clearly shows a decrease in activity at higher concentrations. The inhibition curve for compounds one, five, six, seven, and eight. Shows half maximal inhibitory concentrations at 1.7 micromolar, 21.4 micromolar, 58.8 micromolar, 239.0 micromolar, and greater than 500 micromolar, respectively.
Remember to prepare, the M-P-P-A solution. Just before the beginning of the assay.
