Table 1.
The components of enzyme inhibitory assays consist of types and quantities of enzymes, substrates, indicators, extracts, and detection wavelengths.
| Assay | Assay Components | ||||
|---|---|---|---|---|---|
| Enzyme | Substrate | Indicator | Extract | Detection Wavelength | |
| Lipase | 100 µL of 20 µg/mL lipase 1 | 50 µL of 0.2 mM DMPTB | 10 µL of 16 mM DTNB | 40 µL | 412 nm |
| AChE | 100 µL of 0.25 µg/mL AChE 2 | 50 µL of 0.32 mM ACh | |||
| BChE | 100 µL of 1.5 µg/mL BChE 3 | 50 µL of 0.4 mM BCh | |||
| ACE | 3 µL of 0.5 U/mL ACE 4 | 30 µL of 3 mM HHL | 15 µL of 20 mg/mL o-PDA | 50 µL |
λex = 360 nm λem = 485 nm |
| DPP-IV | 50 µL of 0.02 U/mL DPP-IV 5 | 25 µL of 12 mM Gly-Pro-pNA + 100 µL Tris-HCl (pH 8) | 25 µL | 405 nm | |
| α-Amylase | 100 µL of 0.06 mg/mL α-amylase 6 | 50 µL of 1 mM pCNM | 50 µL | ||
| α-Glucosidase | 10 µL of 0.2 U/mL α-glucosidase 7 | 25 µL of 10 mM pNPG + 160 µL KPB (pH 7) | 5 µL | ||
| BACE-1 | BACE-1 FRET assay kit (Sigma-Aldrich, St. Louis, MO, USA) following manufacturer’s recommendations |
λex = 320 nm λem = 405 nm |
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1Candida rugosa lipase (type VII, ≥ 700 unit/mg); 2Electrophorus electricus AChE (1000 units/mg); 3 equine serum BChE (≥ 10 units/mg); 4 rabbit lung ACE (≥ 2 unit/mg); 5 recombinant human dipeptidyl peptidase-IV (≥ 10 units/mg); 6 porcine pancreatic α-amylase (type VII, ≥ 10 unit/mg); 7Saccharomyces cerevisiae α-glucosidase (type I, ≥ 10 U/mg protein). ACh, acetylthiocholine; AChE, acetycholinesterase; BACE-1, β-secretase; BCh, butyrylthiocholine; BChE, butyrylcholinesterase; pCNM, 2-chloro-4-nitrophenyl-α-D-maltotrioside; DMPTB, 2,3-dimercapto-1-propanol tributyrate; DPP-IV, dipeptidyl peptidase-IV; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); FRET, fluorescence resonance energy transfer; HHL, hippuryl-histidyl-leucine; KPB, potassium phosphate buffer; pNPG, p-nitrophenyl-α-D-glucopyranoside; o-PDA, o-phthaldialdehyde.