NTP hydrolysis

Protocol for NTPase activity assay

• Prepare the following stock solutions:
  • 200 mM MgCl₂
  • 100 mM NTP, stored at -20 ºC in small aliquots to avoid repeated freeze/thaw cycles
• Thaw NTP stock solutions on ice
  • Dilute to 20 mM and 5 mM NTP using water:
• In 1.5mL microcentrifuge tubes, make the following reactions to a final volume of 50 µL
  o Final buffer composition (for SpoIVA): 400 mM NaCl 50mM Tris at pH 7.5, 5 mM MgCl2
  o Add limiting quantity of protein to achieve saturation kinetics (0.3 µM final concentration for SpoIVA, experimentally determined).
  o Add NTP to each tube to achieve the following varying final concentrations (mM): 0, 0.125, 0.250, 0.500, 1.00, 2.00, 4.00 (note: add NTPs last and mix quickly to initiate the reaction). 
  o Adjust final volume to 50 µl with water
• Incubate reactions at 37 ºC for 60 min.
• While reaction is proceeding, remove Malachite Green Phosphate Assay kit (BioAssay Systems, catalog #POMG-25H) from the refrigerator and let it warm to room temperature.
• While reaction is proceeding, use the stock solution of 1 mM phosphate diluted with water to make phosphate solutions at the following concentrations (µM): 0, 4, 8, 12, 16, 24, 32, 40.
• Add 80 µL of each standard to first column of 96-well flat-bottom plate.
• After the reaction has proceeded for 60 min, stop the reaction by diluting each reaction with 950 µl water (20× dilution)
  • Add 80 µL of each reaction to 96-well plate
• Pipette 20 µL Working Reagent (1 ml Reagent A + 10 µl Reagent B) to each well. Using a repeating pipettor will help.
  • Mix by tapping the side of the plate or pipetting up and down.
  • Remove air bubbles using the pipette tip
• Incubate at room temperature for 30 min. The color will develop in each well during this time.
  • Turn on the microplate reader.
• Measure the end point absorbance of each well at 620 nm wavelength

Calculation of k_cat

• Plot a standard curve using the known phosphate standards (absorbance on the y-axis; phosphate concentration on the x-axis). Fit the data to a linear curve using software to obtain an equation that conforms to y = mx + b, where y and x are the values along the y- and x- axes, respectively; m is the slope of the line; and b is the y-intercept.
• Calculate the amount of phosphate released by each reaction using the equation obtained from the standard curve (solving for x), using the observed y value (absorbance at 620 nm) measured for each reaction, and calculated values for m (slope) and b (y-intercept). Correct for the dilution factor (when the reaction was stopped) by multiplying this value by 20. Express this quantity, in the case of SpoIVA, in pmols.
• Divide the value for pmols phosphate released for each reaction by 60 min (the time for each reaction) to obtain the pmols phosphate released by each reaction per minute.
• To obtain the turnover rate (k_cat), divide the value of pmols phosphate released per minute by the protein concentration (in the case of SpoIVA, 0.3 µM final concentration was used for each reaction).
• To calculate K_m, plot the calculated k_cat value for each reaction on the y-axis against the nucleotide concentration used for each reaction on the x-axis. This results in a saturation curve that may be fitted to the Michaelis-Menten enzyme saturation model to obtain K_m.
• Catalytic efficiency is calculated by dividing k_cat by K_m.

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