In Vitro Measurement of α-Galactosidase A and Acid α-Glucosidase Enzyme Activity

1. Cell Harvest and α-galactosidase A or Acid α-glucosidase Activity Measurement

1. Cell harvest​
   1. On the day of the harvest, remove the cells from the incubator and aspirate the medium. Carefully wash the cells 2 times with PBS with Ca₂+ and Mg₂+.
      NOTE: This step is critical because DGJ and DNJ are potent inhibitors of α-galactosidase and α-glucosidase, respectively, and any leftover would invalidate the test.
   2. Add 200 µL of deionized water directly on top of the cells. Rinse the cells from the plate and transfer them to a 1.5 mL reaction tube.

2. Homogenization by freezing and thawing

1. Put the samples in an appropriate foam rack and vortex for 5 s to make the lysis more efficient. Put the samples alternating in liquid nitrogen for 10 s and in a room-temperature water bath until the thawing was complete (5 min).
2. Repeat this procedure 5 times and then spin the samples for 5 min at 10,000 x g. Retain the supernatant and pipette in a new reaction tube.

3. Protein concentration determination using bicinchoninic acid (BCA) assay

1. Prepare a fresh tube for each sample containing 40 µL of deionized H₂O and add 10 µL of the sample. Mix solution by vortexing briefly and transfer 10 µL into a cavity of a 96 well plate (each sample in triplicate). Dilute a 2 mg/mL bovine serum albumin (BSA) stock solution in deionized H₂O as follows to obtain a standard curve: 50 µL H₂O/50 µL BSA; 60 µL H₂O/40 µL BSA; 70 µL H₂O/30 µL BSA; 80 µL H₂O/20 µL BSA; 90 µL H₂O/10 µL BSA; 100 µL H₂O.
2. Start the reaction by adding 200 µL of BCA reagent (Reagent A and reagent B mixed at a 50:1 ratio) and incubate for 1 h in the dark at 37 °C under slight agitation on an orbital shaker (300 rpm). Measure the absorbance at 560 nm in a plate reader.
   NOTE: The samples typically contain between 1 and 1.5 µg protein per µL.

4. Enzyme activity measurement with artificial 4-Methylumbelliferyl substrates (4-MUG)

1. Dilute the calculated amount of each sample and pipet into fresh 1.5 mL reaction tubes to obtain 0.05 (for α-galactosidase A) or 0.5 (for acid α-glucosidase) µg protein/µL solutions. Vortex the samples for 5 s again and pipet 10 µL of this dilution into a 96-well plate (each sample in duplicate).
2. Start the reaction by adding 20 µL of the respective substrate solution:
   For α-galactosidase A: 2 mM 4-Methylumbelliferyl-α-D-galactopyranoside (4-MU-gal) in 0.06 M phosphate citrate buffer, pH 4.7.
   For acid α-glucosidase: 2 mM 4-methylumbelliferyl α-D-glucopyranoside (4-MU-glu) in 0.025 M sodium acetate, pH 4.0.
3. Incubate the enzyme reactions for 1 h in the dark at 37 °C under slight agitation on an orbital shaker (300 rpm). Terminate the reaction by the addition of 200 µL of 1.0 M, pH 10.5 adjusted glycine-NaOH buffer.
4. Prepare a standard curve of 4-methylumbelliferone (4-MU) from a 0.01 mg/mL stock as follows:
   100 µL H2O/ no 4-MU; 80 µL H2O/ 20 µL 4-MU; 60 µL H₂O/ 40 µL 4-MU; 40 µL H₂O/ 60 µL 4-MU; 20 µL H₂O/ 80 µL 4-MU; no H₂O/ 100 µL 4-MU, pipet 10 µL of each dilution into the 96 well plate (in duplicates) and add 200 µL of the 1.0 M glycine-NaOH buffer to each well in order to adjust the volume and pH.
5. Measure the enzyme activity in a fluorescence reader equipped with the appropriate filter set and analyze the data using the appropriate software for the fluorescence reader device.
   NOTE: Both 4-MUG substrates are reduced to 4-MU during exposure to α-galactosidase A or acid α-glucosidase. Released 4-MU is a fluorochrome, which can be measured at 360 and 465 nm as the excitation and emission wavelengths, respectively, using a microplate fluorescence reader.