Table 1 Assay conditions for each enzyme activity assay.
ENZYME | Punch diameter | Reaction buffer | Fluorogenic substrate | Additional reagents | Incubation time | Stop buffer |
|---|---|---|---|---|---|---|
alpha-galactosidase A | 1.5 mm | 30 μL citrate-phosphate 0.3 M pH 5.0 | 30 + 30* μL 4 MU-alpha-D-galactopyranoside 4.2 mM | 10 μL N-acetyl-galactosamine 0.25 M | 20 hours | 10% ethylenediamine 1.32 M pH 11.3 |
alpha-iduronidase | 1.5 mm | 10 μL sodium formate 0.2 M pH 2.8 | 20 + 20* μL 4MU-alpha-L-idopyranoside 2 mM | 10 μL D-saccharic acid 1,4-lactone monohydrate 3 mM | 20 hours | 10% ethylenediamine 1.32 M pH 11.3 |
beta-glucosidase | 3.0 mm | 30 μL citrate-phosphate 0.4 M pH 5.2 | 50 + 50* μL 4MU-beta-D-glycopyranoside 20 mM | 40 μL sodium taurodeoxicolate hydrate 0.75% | 20 hours | 10% ethylenediamine 1.32 M pH 11.3 |
alpha-glucosidase | 1.5 mm | 20 μL sodium acetate 0.4 M pH 4.0 and 6.5 | 20 + 20* μL 4MU-alpha-D-glycopyranoside 2.8 mM | 40 μL acarbose 40 μM | 24 hours | 10% ethylenediamine 1.32 M pH 11.3 |
beta-galactosidase | 1.5 mm | 20 μL citrate-phosphate 0.1 M pH 4.4 | 20 + 40* μL 4MU-beta-D-galactopyranoside 0.8 mM | 20 μL sodium chloride 0.9% | 3 hours | 10% ethylenediamine 1.32 M pH 11.3 |